Nucleic acids and corresponding proteins entitled 158P3D2 useful in treatment and detection of cancer

ABSTRACT

A novel gene 158P3D2 and its encoded protein, and variants thereof, are described wherein 158P3D2 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 158P3D2 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 158P3D2 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 158P3D2 can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 10/994,106, filed Nov. 19, 2004, now U.S. Pat. No. 7,811,575, which is a continuation-in-part of U.S. patent application Ser. No. 10/107,532, filed Mar. 25, 2002, now abandoned, which claims priority to U.S. Provisional Patent Application No. 60/283,112, filed Apr. 10, 2001, and U.S. Provisional Patent Application No. 60/286,630, filed Apr. 25, 2001. The contents of the applications listed in this paragraph are fully incorporated by reference herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

Not applicable.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of the sequence listing via the USPTO EFS-WEB server, as authorized and set forth in MPEP §1730 II.B.2(a)(A), is incorporated herein by reference in its entirety for all purposes. The sequence listing is identified on the electronically filed text file as follows:

File Name Date of Creation Size (bytes) 511582006402Seqlist.txt Sep. 9, 2010 718,856 bytes

FIELD OF THE INVENTION

The invention described herein relates to genes and their encoded proteins, termed 158P3D2 and variants thereof, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 158P3D2.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease—second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.

Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or urethras. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.

Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and eight per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (−2.1% per year). Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men (−1.7% per year) while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.

Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about −0.9% per year) while rates have increased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention relates to a gene, designated 158P3D2, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 158P3D2 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of 158P3D2 are provided. The tissue-related profile of 158P3D2 in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 158P3D2 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary to all or part of the 158P3D2 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 158P3D2-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 158P3D2-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 158P3D2 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 158P3D2 genes, mRNAs, or to 158P3D2-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 158P3D2. Recombinant DNA molecules containing 158P3D2 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 158P3D2 gene products are also provided. The invention further provides antibodies that bind to 158P3D2 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments, there is a proviso that the entire nucleic acid sequence of FIG. 2 is not encoded and/or the entire amino acid sequence of FIG. 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of FIG. 2 is encoded and/or the entire amino acid sequence of FIG. 2 is prepared, either of which are in respective human unit dose forms.

The invention further provides methods for detecting the presence and status of 158P3D2 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 158P3D2. A typical embodiment of this invention provides methods for monitoring 158P3D2 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 158P3D2 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 158P3D2 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 158P3D2 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of 158P3D2. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 158P3D2 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.

In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 158P3D2 and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with 158P3D2 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 158P3D2. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 158P3D2 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 158P3D2 production) or a ribozyme effective to lyse 158P3D2 mRNA.

Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X−1” to each position in Tables VIII-XXI and XXII to XLIX to obtain the actual position of the HLA peptides in their parental molecule. For example, if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150−1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.

One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables VIII-XXI and XXII to XLIX collectively, or an oligonucleotide that encodes the HLA peptide. Another embodiment of the invention comprises an HLA peptide that occurs at least once in Tables VIII-XXI and at least once in tables XXII to XLIX, or an oligonucleotide that encodes the HLA peptide.

Another embodiment of the invention is antibody epitopes, which comprise a peptide regions, or an oligonucleotide encoding the peptide region, that has one two, three, four, or five of the following characteristics:

i) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of FIG. 5;

ii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of FIG. 6;

iii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of FIG. 7;

iv) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of FIG. 8; or

v) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of FIG. 9.[!!! The Figure descriptions need to be revise when using this as a template].

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The 158P3D2 SSH sequence of 312 nucleotides.

FIG. 2A) The cDNA and amino acid sequence of 158P3D2 variant 1 clone 158P3D2-BCP-1 (also called “158P3D2 v.1” or “158P3D2 variant 1”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.

FIG. 2B) The cDNA and amino acid sequence of 158P3D2 variant 2A (also called “158P3D2 v.2”) is shown in FIG. 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 117-827 including the stop codon.

FIG. 2C) The cDNA and amino acid sequence of 158P3D2 variant 2B (also called “158P3D2 v.2”) is shown in FIG. 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 2249-2794 including the stop codon.

FIG. 2D) The cDNA and amino acid sequence of 158P3D2 variant 3 (also called “158P3D2 v.3”) is shown in FIG. 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.

FIG. 2E) The cDNA and amino acid sequence of 158P3D2 variant 4 (also called “158P3D2 v.4”) is shown in FIG. 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.

FIG. 2F) The cDNA and amino acid sequence of 158P3D2 variant 5A (also called “158P3D2 v.5”) is shown in FIG. 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1385 including the stop codon.

FIG. 2G) The cDNA and amino acid sequence of 158P3D2 variant 5B (also called “158P3D2 v.5”) is shown in FIG. 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 1289-1834 including the stop codon.

FIG. 2H) The cDNA and amino acid sequence of 158P3D2 variant 6 (also called “158P3D2 v.6”) is shown in FIG. 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.

FIG. 2I) The cDNA and amino acid sequence of 158P3D2 variant 7 (also called “158P3D2 v.7”) is shown in FIG. 2I. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.

FIG. 2J) The cDNA and amino acid sequence of 158P3D2 variant 8 (also called “158P3D2 v.8”) is shown in FIG. 2J. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon.

FIG. 2K) The cDNA and amino acid sequence of 158P3D2 variant 14 (also called “158P3D2 v.14”) is shown in FIG. 2K. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-4246 including the stop codon.

FIG. 2L) The cDNA and amino acid sequence of 158P3D2 variant 15 (also called “158P3D2 v.15”) is shown in FIG. 2L. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-3502 including the stop codon.

FIG. 2M) The cDNA and amino acid sequence of 158P3D2 variant 16 (also called “158P3D2 v.16”) is shown in FIG. 2M. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-6037 including the stop codon.

FIG. 2N) The cDNA and amino acid sequence of 158P3D2 variant 17 (also called “158P3D2 v.17”) is shown in FIG. 2N. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-6175 including the stop codon.

FIG. 2O) The cDNA and amino acid sequence of 158P3D2 variant 18 (also called “158P3D2 v.18”) is shown in FIG. 2O. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 2932-4764 including the stop codon.

FIG. 2P) The cDNA and amino acid sequence of 158P3D2 variant 19 (also called “158P3D2 v.19”) is shown in FIG. 2P. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-6001 including the stop codon.

FIG. 2Q) The cDNA and amino acid sequence of 158P3D2 variant 20 (also called “158P3D2 v.20”) is shown in FIG. 2Q. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 65-6121 including the stop codon.

FIG. 2R) 158P3D2 v.9 through v.13, SNP variants of 158P3D2 v.1. The 158P3D2 v.9 through v.13 proteins have 1072 amino acids. Variants 158P3D2 v.4 through v.20 are variants with single nucleotide difference from 158P3D2 v.1. 158P3D2 v.10, v.12 and v.13 proteins differ from 158P3D2 v.1 by one amino acid. 158P3D2 v.9 and v.11 proteins code for the same protein as v.1. Though these SNP variants are shown separately, they can also occur in any combinations and in any of the transcript variants listed above in FIGS. 2A-2Q.

FIG. 3A) The amino acid sequence of 158P3D2 v.1 clone 158P3D2-BCP-1 is shown in FIG. 3A; it has 328 amino acids.

FIG. 3B) The amino acid sequence of 158P3D2 v.2A is shown in FIG. 3B; it has 236 amino acids.

FIG. 3C) The amino acid sequence of 158P3D2 v.2B is shown in FIG. 3C; it has 181 amino acids.

FIG. 3D) The amino acid sequence of 158P3D2 v.3 is shown in FIG. 3D; it has 328 amino acids.

FIG. 3E) The amino acid sequence of 158P3D2 v.4 is shown in FIG. 3E; it has 328 amino acids.

FIG. 3F) The amino acid sequence of 158P3D2 v.5A is shown in FIG. 3F; it has 178 amino acids.

FIG. 3G) The amino acid sequence of 158P3D2 v.5B is shown in FIG. 3G; it has 181 amino acids.

FIG. 3H) The amino acid sequence of 158P3D2 v.10 is shown in FIG. 3H; it has 328 amino acids.

FIG. 3I) The amino acid sequence of 158P3D2 v.11 is shown in FIG. 3I; it has 328 amino acids.

FIG. 3J) The amino acid sequence of 158P3D2 v.12 is shown in FIG. 3J; it has 328 amino acids.

FIG. 3K) The amino acid sequence of 158P3D2 v.13 is shown in FIG. 3K; it has 328 amino acids.

FIG. 3L) The amino acid sequence of 158P3D2 v.14 is shown in FIG. 3L; it has 1393 amino acids.

FIG. 3M) The amino acid sequence of 158P3D2 v.15 is shown in FIG. 3M; it has 1145 amino acids.

FIG. 3N) The amino acid sequence of 158P3D2 v.16 is shown in FIG. 3N; it has 1990 amino acids.

FIG. 3O) The amino acid sequence of 158P3D2 v.17 is shown in FIG. 3O; it has 2036 amino acids.

FIG. 3P) The amino acid sequence of 158P3D2 v.18 is shown in FIG. 3P; it has 610 amino acids.

FIG. 3Q) The amino acid sequence of 158P3D2 v.19 is shown in FIG. 3Q; it has 1978 amino acids.

FIG. 3R) The amino acid sequence of 158P3D2 v.20 is shown in FIG. 3R; it has 2018 amino acids.

As used herein, a reference to 158P3D2 includes all variants thereof, including those shown in FIGS. 2, 3, 10, 11, and 12 unless the context clearly indicates otherwise.

FIG. 4. Effect of 158P3D2 RNAi on cell proliferation. SCaBER cells or Cos-1 cells were transfected with Lipofectamine 2000 reagent (LF2K) alone, or with negative control Luc4 oligo (20 nM), positive control Eg5 oligo (20 nM) or 158P3D2.b oligo (20 nM). After 48 hours, the media was replaced and the cells were incubated for 24 hrs, pulsed with ³H-thymidine at 1.5 μCi/ml for 14 hrs, harvested onto a filtermat and counted in scintillation cocktail on a Microbeta trilux counter. Percentage cell proliferation relative to the LF2k control (100%) is shown. The reduction in 158P3D2 levels by the 158P3D2.b siRNA oligo correlated with diminished cell proliferation in the SCaBER cells, but no effect was observed in the 158P3D2-negative cell line Cos-1.

FIG. 5( a)-(i). Hydrophilicity amino acid profile of 158P3D2 v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website located on the World Wide Web at (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 6( a)-(i). Hydropathicity amino acid profile of 158P3D2 v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 7( a)-(i). Percent accessible residues amino acid profile of 158P3D2 v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 8( a)-(i). Average flexibility amino acid profile of 158P3D2 v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 9( a)-(i). Beta-turn amino acid profile of 158P3D2 v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.

FIG. 10. Exon compositions of transcript variants of 158P3D2. Variant 158P3D2 v.2, v.14 through v.20 are transcript variants. Compared with 158P3D2 v.1; v.2 had six additional exons to the 5′ end, an exon 7 longer than exon 1 of 158P3D2 v.1 and an exon 10 shorter than exon 4 of 158P3D2 v.1. Exons 2, 3, 5, 6 and 7 of 158P3D2 v.1 are the same as exons 8, 9, 11, 12 and 13 of 158P3D2 v.2, respectively. Other variants had different exon compositions as shown above. Numbers in “( )” underneath the box correspond to those of 158P3D2 v.1. Black boxes show the same sequence as 158P3D2 v.1. Length of introns are not proportional.

FIG. 11. Schematic display of protein variants of 158P3D2. Nucleotide variant 158P3D2 v.2 and 158P3D2 v.5 potentially coded for two different proteins, designated as variants 158P3D2 v.2A and 158P3D2 v.2B, 158P3D2 v.5A and 158P3D2 v.5B, respectively. Variant 158P3D2 v.5B shares the same amino acid sequence as variant 158P3D2 v.2B. Variants 158P3D2 v.3 and v.4 were variants with single amino acid variations. Black box shows the same sequence as 158P3D2 v.1. Numbers in “( )” underneath the black boxes correspond to those of 158P3D2 v.1 and those underneath the “brick” boxes correspond to those of v.17. Single amino acid differences are indicated above the box.

FIG. 12. Schematic display of SNP variants of 158P3D2. Variant 158P3D2 v.3 through v.13 are variants with a single nucleotide difference from v.1. Though these alternative SNP alleles were shown separately, they could occur in any transcript variants in any combination (called haplotype). Numbers in “( )” underneath the box correspond to those of 158P3D2 v.1. ‘-’ indicate single nucleotide deletion. Black boxes show the same sequence as 158P3D2 v.1. SNPs are indicated above the box.

FIG. 13. Secondary structure and transmembrane domains prediction for 158P3D2 protein variants.

FIG. 13A (SEQ ID NO: 54), FIG. 13B (SEQ ID NO: 55), FIG. 13C (SEQ ID NO: 56), FIG. 13D (SEQ ID NO: 57), FIG. 13E (SEQ ID NO: 58), FIG. 13F (SEQ ID NO: 59), FIG. 13G (SEQ ID NO: 60), FIG. 13H (SEQ ID NO: 61), FIG. 13I (SEQ ID NO: 62): The secondary structures of 158P3D2 protein variants 1, 2a, 2b, 5a, 14, 15, 16, 17, 18 respectively, were predicted using the HNN—Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deléage G., accessed from the ExPasy molecular biology server. This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein variant in a given secondary structure is also listed.

FIG. 13J, FIG. 13L, FIG. 13N, FIG. 13P, FIG. 13R, FIG. 13T, FIG. 13V, FIG. 13X, and FIG. 13Z: Schematic representation of the probability of existence of transmembrane regions of 158P3D2 protein variants 1, 2a, 2b, 5a, 14, 15, 16, 17, 18 respectively, based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE-A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). FIG. 13K, FIG. 13M, FIG. 13O, FIG. 13Q, FIG. 13S, FIG. 13U, FIG. 13W, FIG. 13Y, FIG. 13AA: Schematic representation of the probability of the existence of transmembrane regions of 158P3D2 variants 1, 2a, 2b, 5a, 14, 15, 16, 17, 18 respectively, based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server.

FIG. 14. 158P3D2 Expression in Normal and Cancer Tissue Specimens. First strand cDNA was prepared from a panel of 13 normal tissues (brain, heart, kidney, liver, lung, spleen, skeletal muscle, testis, pancreas, colon, stomach) and pools of 4-7 patients from the following cancer indications: bladder, kidney, colon, lung, pancreas, stomach, ovary, breast, multiple cancer metastasis, cervix, lymphoma as well as from a pool of patient-derived xenografts (prostate cancer, bladder cancer and kidney cancer). Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the AlphaImager software. Results show strong expression of 158P3D2 in cancers of the bladder, kidney, colon, lung, pancreas, stomach, ovary, breast, cervix, and lymphoma. Low expression was detected in all normal tissues tested except in normal stomach. Strong expression was also observed in the cancer metastasis pool.

FIG. 15. 158P3D2 Expression in bladder cancer patient specimens. First strand cDNA was prepared from normal bladder, bladder cancer cell lines (UM-UC-3, TCCSUP, J82) and a panel of bladder cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in the majority of bladder cancer patient specimens tested. Very low expression was detected in normal tissues, but no expression was seen in the cell lines tested.

FIG. 16. 158P3D2 Expression in bladder cancer patient specimens by northern blotting. RNA was extracted from normal bladder, bladder cancer cell lines (UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) and their normal adjacent tissues (NAT). Northern blot with 10 ug of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor tissues, but not in normal nor NAT tissues.

FIG. 17. 158P3D2 Expression in lung cancer patient specimens. First strand cDNA was prepared from normal lung, cancer cell line A427 and a panel of lung cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in 35/39 (90%) of lung cancer specimens, but not in all 3 normal lung tissues tested.

FIG. 18. 158P3D2 Expression in lung cancer patient specimens by northern blotting. RNA was extracted from normal lung, A427 lung cancer cell line, and a panel of lung cancer patient specimens. Northern blot with 10 ug of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in normal tissues.

FIG. 19. 158P3D2 Expression in cancer metastasis patient specimens. First strand cDNA was prepared from normal colon, kidney, liver, lung, pancreas, stomach and from a panel of cancer metastasis patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in the majority of patient cancer metastasis specimens tested but not in normal tissues.

FIG. 20. 158P3D2 Expression in cervical cancer patient specimens. First strand cDNA was prepared from normal cervix, cervical cancer cell line HeLa, and a panel of cervical cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in all 14 cervical cancer patient specimens tested. No expression was detected in normal cervix nor in the cell line tested.

FIG. 21. 158P3D2 Expression in cervical cancer patient specimens by northern blotting. RNA was extracted from normal cervix, cervical cancer cell line HeLa, and a panel of cervical cancer patient specimens. Northern blot with 10 ug of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor tissues, but not in normal cervix nor in the cell line.

FIG. 22. 158P3D2 Expression in kidney cancer patient specimens. First strand cDNA was prepared from normal kidney, kidney cancer cell lines (769-P, A-498, CAKI-1), and a panel of kidney cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of kidney cancer patient specimens, but not in all 3 normal kidney tissues tested. Low expression was detected in 2 of 3 cell lines tested.

FIG. 23. 158P3D2 Expression in kidney cancer patient specimens by northern blotting. RNA was extracted from normal kidney and a panel of kidney cancer patient specimens. Northern blot with 10 ug of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in the normal tissue.

FIG. 24. 158P3D2 Expression in stomach cancer patient specimens. First strand cDNA was prepared from normal stomach, and a panel of stomach cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of stomach cancer patient specimens. Weak expression was detected in the 2 normal stomach, and only in 1 of the 2 NAT tissues tested.

FIG. 25. 158P3D2 Expression in stomach cancer patient specimens by northern blotting. RNA was extracted from normal stomach and a panel of stomach cancer patient specimens. Northern blot with 10 ug of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in the normal tissue.

FIG. 26. 158P3D2 Expression in colon cancer patient specimens. First strand cDNA was prepared from normal colon, colon cancer cell lines (LoVo, CaCO-2, SK CO 1, Colo 205, T284), and a panel of colon cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of colon cancer patient specimens. But it was weakly expressed in just 2 of 3 normal tissues, and 3 of 5 cell lines tested.

FIG. 27. 158P3D2 Expression in uterus cancer patient specimens. First strand cDNA was prepared from normal uterus and a panel of uterus cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show 158P3D2 is expressed at varying levels in the majority of uterus cancer patient specimens, but not in normal uterus.

FIG. 28. 158P3D2 Expression in breast cancer patient specimens. First strand cDNA was prepared from normal breast, breast cancer cell lines (MD-MBA-435S, DU4475, MCF-7, CAMA-1, MCF10A), and a panel of breast cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show 158P3D2 is expressed at varying levels in the majority of breast cancer patient specimens. But it was weakly expressed in just 2 of 3 normal tissues, and 2 of 5 cell lines tested.

FIG. 29. Serum titer of mice immunized with KLH-peptide encoding amino acids 315-328 of 158P3D2. Serial dilutions of serum taken from immunized mice were incubated on an ELISA plate coated with the 158P3 D2 peptide conjugated to ovalbumin. Specific bound antibody was then detected by incubation goat anti-mouse IgG-HRP conjugate and then visualized and quantitated by development with TMB substrate and optical density determination.

FIG. 30. Validation of 158P3D2 siRNA oligo. Cos-1 cells were transfected with 1 μg pcDNA3-158P3D2, which encodes a full-length 158P3D2 protein fusion with a Myc/His tag on the C-terminus, simultaneously with Lipofectamine 2000 reagent (LF2k) alone, or with control CT1 oligo (20 nM), 158P3D2.b oligo (20 nM), or no DNA or oligo (No DNA). After 72 hours, the cells were lysed in 1% Triton buffer, and 50 μg of total soluble cell lysate was analyzed by Western blotting. The upper panel was blotted with anti-Myc (1:1000) to detect the 158P3D2-Myc/His fusion protein and the lower panel was developed with anti-actin. The level of 158P3D2 was diminished by the 158P3D2 siRNA oligo, whereas no change was observed with the control (LF2k) or siRNA oligo CT1. In contrast, no change in the level of actin was noted in the cell lysates, indicating that the loading was equivalent in all lanes.

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections

-   I.) Definitions -   II.) 158P3D2 Polynucleotides -   II.A.) Uses of 158P3D2 Polynucleotides -   II.A.1.) Monitoring of Genetic Abnormalities -   II.A.2.) Antisense Embodiments -   II.A.3.) Primers and Primer Pairs -   II.A.4.) Isolation of 158P3D2-Encoding Nucleic Acid Molecules -   II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems -   III.) 158P3D2-related Proteins -   III.A.) Motif-bearing Protein Embodiments -   III.B.) Expression of 158P3D2-related Proteins -   III.C.) Modifications of 158P3D2-related Proteins -   III.D.) Uses of 158P3D2-related Proteins -   IV.) 158P3D2 Antibodies -   V.) 158P3D2 Cellular Immune Responses -   VI.) 158P3D2 Transgenic Animals -   VII.) Methods for the Detection of 158P3D2 -   VIII.) Methods for Monitoring the Status of 158P3D2-related Genes     and Their Products -   IX.) Identification of Molecules That Interact With 158P3D2 -   X.) Therapeutic Methods and Compositions -   X.A.) Anti-Cancer Vaccines -   X.B.) 158P3D2 as a Target for Antibody-Based Therapy -   X.C.) 158P3D2 as a Target for Cellular Immune Responses -   X.C.1. Minigene Vaccines -   X.C.2. Combinations of CTL Peptides with Helper Peptides -   X.C.3. Combinations of CTL Peptides with T Cell Priming Agents -   X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL     Peptides -   X.D.) Adoptive Immunotherapy -   X.E.) Administration of Vaccines for Therapeutic or Prophylactic     Purposes -   XI.) Diagnostic and Prognostic Embodiments of 158P3D2. -   XII.) Inhibition of 158P3D2 Protein Function -   XII.A.) Inhibition of 158P3D2 With Intracellular Antibodies -   XII.B.) Inhibition of 158P3D2 with Recombinant Proteins -   XII.C.) Inhibition of 158P3D2 Transcription or Translation -   XII.D.) General Considerations for Therapeutic Strategies -   XIII.) Identification, Characterization and Use of Modulators of     109P1D1 -   XIII.A.) Methods to Identify and Use Modulators -   XIII.B.) Gene Expression-related Assays -   XIII.C.) Expression Monitoring to Identify Compounds that Modify     Gene Expression -   XIII.D.) Biological Activity-related Assays -   XIII.E.) High Throughput Screening to Identify Modulators -   XIII.F.) Use of Soft Agar Growth and Colony Formation to Identify     and Characterize Modulators -   XIII.G.) Evaluation of Contact Inhibition and Growth Density     Limitation to Identify and Characterize Modulators -   XIII.H.) Evaluation of Growth Factor or Serum Dependence to Identify     and Characterize Modulators -   XIII.I.) Use of Tumor-specific Marker Levels to Identify and     Characterize Modulators -   XIII.J.) Invasiveness into Matrigel to Identify and Characterize     Modulators -   XIII.K.) Evaluation of Tumor Growth In Vivo to Identify and     Characterize Modulators -   XIII.L.) In Vitro Assays to Identify and Characterize Modulators -   XIII.M.) Binding Assays to Identify and Characterize Modulators -   XIII.N.) Competitive Binding to Identify and Characterize Modulators -   XIII.O.) Use of Polynucleotides to Down-regulate or Inhibit a     Protein of the Invention. -   XIII.P.) Inhibitory and Antisense Nucleotides -   XIII.Q.) Ribozymes -   XIII.R.) Use of Modulators in Phenotypic Screening -   XIII.S.) Use of Modulators to Affect Peptides of the Invention -   XIII.T.) Methods of Identifying Characterizing Cancer-associated     Sequences -   XIV.) KITS/Articles of Manufacture

I.) DEFINITIONS

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.

“Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 158P3D2 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 158P3D2. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 158P3D2-related protein). For example, an analog of a 158P3D2 protein can be specifically bound by an antibody or T cell that specifically binds to 158P3D2.

The term “antibody” is used in the broadest sense. Therefore, an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-158P3D2 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-158P3D2 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-158P3D2 antibody compositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an “expression enhanced sequences.”

A “combinatorial library” is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Numerous chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)).

Preparation and screening of combinatorial libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbarnates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g., Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), and U.S. Pat. No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A, Applied Biosystems, Foster City, Calif.; 9050, Plus, Millipore, Bedford, NIA). A number of well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations such as the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).

The term “cytotoxic agent” refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32 and radioactive isotopes of Lu including Lu177. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.

The “gene product” is sometimes referred to herein as a protein or mRNA. For example, a “gene product of the invention” is sometimes referred to herein as a “cancer amino acid sequence”, “cancer protein”, “protein of a cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listed in Table I”, etc. In one embodiment, the cancer protein is encoded by a nucleic acid of FIG. 2. The cancer protein can be a fragment, or alternatively, be the full-length protein to the fragment encoded by the nucleic acids of FIG. 2. In one embodiment, a cancer amino acid sequence is used to determine sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of FIG. 2. In another embodiment, the sequences are sequence variants as further described herein.

“High throughput screening” assays for the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Pat. No. 5,559,410 discloses high throughput screening methods for proteins; U.S. Pat. No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays); while U.S. Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.

In addition, high throughput screening systems are commercially available (see, e.g., Amersham Biosciences, Piscataway, N.J.; Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass.; etc.). These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.

The term “homolog” refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., Immunology, 8th Ed., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C. and temperatures for washing in 0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be “isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 158P3D2 genes or that encode polypeptides other than 158P3D2 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 158P3D2 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 158P3D2 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 158P3D2 protein. Alternatively, an isolated protein can be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage T×N×M+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.

The term “modulator” or “test compound” or “drug candidate” or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, e.g., a nucleic acid or protein sequences, or effects of cancer sequences (e.g., signaling, gene expression, protein interaction, etc.) In one aspect, a modulator will neutralize the effect of a cancer protein of the invention. By “neutralize” is meant that an activity of a protein is inhibited or blocked, along with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect of a gene, and its corresponding protein, of the invention by normalizing levels of said protein. In preferred embodiments, modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein, or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint. In another embodiment, a modulator induced a cancer phenotype. Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.

Modulators, drug candidates or test compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also comprise biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. One class of modulators are peptides, for example of from about five to about 35 amino acids, with from about five to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulatory protein is soluble, includes a non-transmembrane region, and/or, has an N-terminal Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid in coupling, i.e., to cysteine. In one embodiment, a cancer protein of the invention is conjugated to an immunogenic agent as discussed herein. In one embodiment, the cancer protein is conjugated to BSA. The peptides of the invention, e.g., of preferred lengths, can be linked to each other or to other amino acids to create a longer peptide/protein. The modulatory peptides can be digests of naturally occurring proteins as is outlined above, random peptides, or “biased” random peptides. In a preferred embodiment, peptide/protein-based modulators are antibodies, and fragments thereof, as defined herein.

Modulators of cancer can also be nucleic acids. Nucleic acid modulating agents can be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be used in an approach analogous to that outlined above for proteins.

The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.

A “motif”, as in biological motif of a 158P3D2-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.

The term “polynucleotide” means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with “oligonucleotide”. A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T), as shown for example in FIG. 2, can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA class I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention. Alternatively, in another embodiment, the primary anchor residues of a peptide binds an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.

“Radioisotopes” include, but are not limited to the following (non-limiting exemplary uses are also set forth):

Examples of Medical Isotopes

Isotope Description of use Actinium-225 See Thorium-229 (Th-229) (AC-225) Actinium-227 Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat (AC-227) metastases in the skeleton resulting from cancer (i.e., breast and prostate cancers), and cancer radioimmunotherapy Bismuth-212 See Thorium-228 (Th-228) (Bi-212) Bismuth-213 See Thorium-229 (Th-229) (Bi-213) Cadmium-109 Cancer detection (Cd-109) Cobalt-60 Radiation source for radiotherapy of cancer, for food irradiators, and for (Co-60) sterilization of medical supplies Copper-64 A positron emitter used for cancer therapy and SPECT imaging (Cu-64) Copper-67 Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic (Cu-67) studies (i.e., breast and colon cancers, and lymphoma) Dysprosium-166 Cancer radioimmunotherapy (Dy-166) Erbium-169 Rheumatoid arthritis treatment, particularly for the small joints associated (Er-169) with fingers and toes Europium-152 Radiation source for food irradiation and for sterilization of medical (Eu-152) supplies Europium-154 Radiation source for food irradiation and for sterilization of medical (Eu-154) supplies Gadolinium-153 Osteoporosis detection and nuclear medical quality assurance devices (Gd-153) Gold-198 Implant and intracavity therapy of ovarian, prostate, and brain cancers (Au-198) Holmium-166 Multiple myeloma treatment in targeted skeletal therapy, cancer (Ho-166) radioimmunotherapy, bone marrow ablation, and rheumatoid arthritis treatment Iodine-125 Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer (I-125) treatment, radiolabeling, tumor imaging, mapping of receptors in the brain, interstitial radiation therapy, brachytherapy for treatment of prostate cancer, determination of glomerular filtration rate (GFR), determination of plasma volume, detection of deep vein thrombosis of the legs Iodine-131 Thyroid function evaluation, thyroid disease detection, treatment of thyroid (I-131) cancer as well as other non-malignant thyroid diseases (i.e., Graves disease, goiters, and hyperthyroidism), treatment of leukemia, lymphoma, and other forms of cancer (e.g., breast cancer) using radioimmunotherapy Iridium-192 Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked (Ir-192) arteries (i.e., arteriosclerosis and restenosis), and implants for breast and prostate tumors Lutetium-177 Cancer radioimmunotherapy and treatment of blocked arteries (i.e., (Lu-177) arteriosclerosis and restenosis) Molybdenum-99 Parent of Technetium-99m (Tc-99m) which is used for imaging the brain, (Mo-99) liver, lungs, heart, and other organs. Currently, Tc-99m is the most widely used radioisotope used for diagnostic imaging of various cancers and diseases involving the brain, heart, liver, lungs; also used in detection of deep vein thrombosis of the legs Osmium-194 Cancer radioimmunotherapy (Os-194) Palladium-103 Prostate cancer treatment (Pd-103) Platinum-195m Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic (Pt-195m) drug Phosphorus-32 Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone (P-32) cancer diagnosis/treatment; colon, pancreatic, and liver cancer treatment; radiolabeling nucleic acids for in vitro research, diagnosis of superficial tumors, treatment of blocked arteries (i.e., arteriosclerosis and restenosis), and intracavity therapy Phosphorus-33 Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, and (P-33) treatment of blocked arteries (i.e., arteriosclerosis and restenosis) Radium-223 See Actinium-227 (Ac-227) (Ra-223) Rhenium-186 Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and (Re-186) treatment of lymphoma and bone, breast, colon, and liver cancers using radioimmunotherapy Rhenium-188 Cancer diagnosis and treatment using radioimmunotherapy, bone cancer (Re-188) pain relief, treatment of rheumatoid arthritis, and treatment of prostate cancer Rhodium-105 Cancer radioimmunotherapy (Rh-105) Samarium-145 Ocular cancer treatment (Sm-145) Samarium-153 Cancer radioimmunotherapy and bone cancer pain relief (Sm-153) Scandium-47 Cancer radioimmunotherapy and bone cancer pain relief (Sc-47) Selenium-75 Radiotracer used in brain studies, imaging of adrenal cortex by gamma- (Se-75) scintigraphy, lateral locations of steroid secreting tumors, pancreatic scanning, detection of hyperactive parathyroid glands, measure rate of bile acid loss from the endogenous pool Strontium-85 Bone cancer detection and brain scans (Sr-85) Strontium-89 Bone cancer pain relief, multiple myeloma treatment, and osteoblastic (Sr-89) therapy Technetium-99m See Molybdenum-99 (Mo-99) (Tc-99m) Thorium-228 Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in cancer (Th-228) radioimmunotherapy Thorium-229 Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213 (Bi-213) (Th-229) which are alpha emitters used in cancer radioimmunotherapy Thulium-170 Gamma source for blood irradiators, energy source for implanted medical (Tm-170) devices Tin-117m Cancer immunotherapy and bone cancer pain relief (Sn-117m) Tungsten-188 Parent for Rhenium-188 (Re-188) which is used for cancer (W-188) diagnostics/treatment, bone cancer pain relief, rheumatoid arthritis treatment, and treatment of blocked arteries (i.e., arteriosclerosis and restenosis) Xenon-127 Neuroimaging of brain disorders, high resolution SPECT studies, (Xe-127) pulmonary function tests, and cerebral blood flow studies Ytterbium-175 Cancer radioimmunotherapy (Yb-175) Yttrium-90 Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancer (Y-90) treatment Yttrium-91 A gamma-emitting label for Yttrium-90 (Y-90) which is used for cancer (Y-91) radioimmunotherapy (i.e., lymphoma, breast, colon, kidney, lung, ovarian, prostate, pancreatic, and inoperable liver cancers)

By “randomized” or grammatical equivalents as herein applied to nucleic acids and proteins is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides (or nucleic acids, discussed herein) can incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.

In one embodiment, a library is “fully randomized,” with no sequence preferences or constants at any position. In another embodiment, the library is a “biased random” library. That is, some positions within the sequence either are held constant, or are selected from a limited number of possibilities. For example, the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind or interact with 158P3D2, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 158P3D2 protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules physically associate with, or bind, 158P3D2 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions.

“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. “Moderately stringent conditions” are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

An HLA “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Overall phenotypic frequencies of HLA-supertypes in different ethnic populations are set forth in Table IV (F). The non-limiting constituents of various supetypes are as follows:

-   -   A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802,         A*6901, A*0207     -   A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101     -   B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601,         B*6701, B*7801, B*0702, B*5101, B*5602     -   B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)     -   A1: A*0102, A*2604, A*3601, A*4301, A*8001     -   A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003     -   B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02,         B*3901, B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08     -   B58: B*1516, B*1517, B*5701, B*5702, B58     -   B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)

Calculated population coverage afforded by different HLA-supertype combinations are set forth in Table IV (G).

As used herein “to treat” or “therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; full eradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A “transgene” is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The “one or more peptides” can include any whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 158P3D2 protein shown in FIG. 2 or FIG. 3. An analog is an example of a variant protein. Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.

The “158P3D2-related proteins” of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 158P3D2 proteins or fragments thereof, as well as fusion proteins of a 158P3D2 protein and a heterologous polypeptide are also included. Such 158P3D2 proteins are collectively referred to as the 158P3D2-related proteins, the proteins of the invention, or 158P3D2. The term “158P3D2-related protein” refers to a polypeptide fragment or a 158P3D2 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 576 or more amino acids.

II.) 158P3D2 POLYNUCLEOTIDES

One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 158P3D2 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 158P3D2-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 158P3D2 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 158P3D2 gene, mRNA, or to a 158P3D2 encoding polynucleotide (collectively, “158P3D2 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.

Embodiments of a 158P3D2 polynucleotide include: a 158P3D2 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 158P3D2 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 158P3D2 nucleotides comprise, without limitation:

(I) a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in FIG. 2, wherein T can also be U;

(II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2A, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;

(III) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2B, from nucleotide residue number 117 through nucleotide residue number 827, including the stop codon, wherein T can also be U;

(IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2C, from nucleotide residue number 2249 through nucleotide residue number 2794, including the a stop codon, wherein T can also be U;

(V) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2D, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;

(VI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2E, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;

(VII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2F, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;

(VIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2G, from nucleotide residue number 1289 through nucleotide residue number 1834, including the stop codon, wherein T can also be U;

(IX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2H, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;

(X) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2I, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;

(XI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2J, from nucleotide residue number 849 through nucleotide residue number 1835, including the stop codon, wherein T can also be U;

(XII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2K, from nucleotide residue number 65 through nucleotide residue number 4246, including the stop codon, wherein T can also be U;

(XIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2L, from nucleotide residue number 65 through nucleotide residue number 3502, including the stop codon, wherein T can also be U;

(XIV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2M, from nucleotide residue number 65 through nucleotide residue number 6037, including the stop codon, wherein T can also be U;

(XV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2N, from nucleotide residue number 65 through nucleotide residue number 6175, including the stop codon, wherein T can also be U;

(XVI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2O, from nucleotide residue number 2932 through nucleotide residue number 4764, including the stop codon, wherein T can also be U;

(XVII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2P, from nucleotide residue number 65 through nucleotide residue number 6001, including the stop codon, wherein T can also be U;

(XVIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2Q, from nucleotide residue number 65 through nucleotide residue number 6121, including the stop codon, wherein T can also be U;

(XIX) a polynucleotide that encodes a 158P3D2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIG. 2A-Q;

(XX) a polynucleotide that encodes a 158P3D2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIG. 2A-Q;

(XXI) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX;

(XXII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A-3R in any whole number increment up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610, 1978, and 2018 that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(XXIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A-3R in any whole number increment up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610, 1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XXIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A-3R in any whole number increment up to 32.8, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610, 1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XXV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3A-3R in any whole number increment up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610, 1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XXVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3A-3R in any whole number increment up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610, 1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XXVII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XXVI);

(XXVIII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XXVII);

(XXIX) a peptide that is encoded by any of (I) to (XXVIII); and;

(XXX) a composition comprising a polynucleotide of any of (I)-(XXVIII) or peptide of (XXIX) together with a pharmaceutical excipient and/or in a human unit dose form;

(XXXI) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to modulate a cell expressing 158P3D2;

(XXXII) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 158P3D2;

(XXIII) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 158P3D2, said cell from a cancer of a tissue listed in Table I;

(XXXIV) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat a cancer;

(XXXV) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat a cancer of a tissue listed in Table I; and;

(XXXVI) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to identify or characterize a modulator of a cell expressing 158P3D2.

As used herein, a range is understood to disclose specifically all whole unit positions thereof.

Typical embodiments of the invention disclosed herein include 158P3D2 polynucleotides that encode specific portions of 158P3D2 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325 and 328 or more contiguous amino acids of 158P3D2 variant 1; the maximal lengths relevant for other variants are shown in FIGS. 2A-2Q and 3A-3R respectively.

For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3. Accordingly, polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids, 100 through the carboxyl terminal amino acid of the 158P3D2 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a 158P3D2 protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 158P3D2 protein “or variant” shown in FIG. 2 or FIG. 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 158P3D2 sequence as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed herein include 158P3D2 polynucleotide fragments encoding one or more of the biological motifs contained within a 158P3D2 protein “or variant” sequence, including one or more of the motif-bearing subsequences of a 158P3D2 protein “or variant” set forth in Tables VIII-XXI and XXII-XLIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 158P3D2 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 158P3D2 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.

Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and Tables XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides listed in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X minus 1” to each position in Tables VIII-XXI and Tables XXII-IL to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150−1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.

II.A.) Uses of 158P3D2 Polynucleotides

II.A.1. Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 158P3D2 gene maps to the chromosomal location set forth in the Example entitled “Chromosomal Mapping of 158P3D2.” For example, because the 158P3D2 gene maps to this chromosome, polynucleotides that encode different regions of the 158P3D2 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 158P3D2 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 158P3D2 that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 158P3D2 was shown to be highly expressed in prostate and other cancers, 158P3D2 polynucleotides are used in methods assessing the status of 158P3D2 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 158P3D2 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 158P3D2 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.

II.A.2. Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 158P3D2. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 158P3D2 polynucleotides and polynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term “antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 158P3D2. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 158P3D2 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 158P3D2 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).

The 158P3D2 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5′ codons or last 100 3′ codons of a 158P3D2 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 158P3D2 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 158P3D2 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 158P3D2 mRNA. Optionally, 158P3D2 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5′ codons or last 10 3′ codons of 158P3D2. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 158P3D2 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3. Primers and Primer Pairs

Further specific embodiments of these nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 158P3D2 polynucleotide in a sample and as a means for detecting a cell expressing a 158P3D2 protein.

Examples of such probes include polypeptides comprising all or part of the human 158P3D2 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 158P3D2 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 158P3D2 mRNA.

The 158P3D2 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 158P3D2 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 158P3D2 polypeptides; as tools for modulating or inhibiting the expression of the 158P3D2 gene(s) and/or translation of the 158P3D2 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described herein to identify and isolate a 158P3D2 or 158P3D2 related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence per se, which would comprise all or most of the sequences found in the probe used.

II.A.4. Isolation of 158P3D2-Encoding Nucleic Acid Molecules

The 158P3D2 cDNA sequences described herein enable the isolation of other polynucleotides encoding 158P3D2 gene product(s), as well as the isolation of polynucleotides encoding 158P3D2 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 158P3D2 gene product as well as polynucleotides that encode analogs of 158P3D2-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 158P3D2 gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 158P3D2 gene cDNAs can be identified by probing with a labeled 158P3D2 cDNA or a fragment thereof. For example, in one embodiment, a 158P3D2 cDNA (e.g., FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 158P3D2 gene. A 158P3D2 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 158P3D2 DNA probes or primers.

II.A.5. Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containing a 158P3D2 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 158P3D2 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPr1, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 158P3D2 or a fragment, analog or homolog thereof can be used to generate 158P3D2 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of 158P3D2 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 158P3D2 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPr1. The host-vector systems of the invention are useful for the production of a 158P3D2 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 158P3D2 and 158P3D2 mutations or analogs.

Recombinant human 158P3D2 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 158P3D2-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 158P3D2 or fragment, analog or homolog thereof, a 158P3D2-related protein is expressed in the 293T cells, and the recombinant 158P3D2 protein is isolated using standard purification methods (e.g., affinity purification using anti-158P3D2 antibodies). In another embodiment, a 158P3D2 coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish 158P3D2 expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a 158P3D2 coding sequence can be used for the generation of a secreted form of recombinant 158P3D2 protein.

As discussed herein, redundancy in the genetic code permits variation in 158P3D2 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as at URL dna.affrc.go.jp/˜nakamura/codon.html.

Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5′ proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 158P3D2-RELATED PROTEINS

Another aspect of the present invention provides 158P3D2-related proteins. Specific embodiments of 158P3D2 proteins comprise a polypeptide having all or part of the amino acid sequence of human 158P3D2 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 158P3D2 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 158P3D2 shown in FIG. 2 or FIG. 3.

Embodiments of a 158P3D2 polypeptide include: a 158P3D2 polypeptide having a sequence shown in FIG. 2, a peptide sequence of a 158P3D2 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in FIG. 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 158P3D2 peptides comprise, without limitation:

(I) a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in FIG. 2A-Q or FIG. 3A-3R;

(II) a 158P3D2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIG. 2A-Q or 3A-R;

(III) a 158P3D2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIG. 2A-Q or 3A-R;

(IV) a protein that comprises at least one peptide set forth in Tables VIII to XLIX, optionally with a proviso that it is not an entire protein of FIG. 2;

(V) a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is also set forth in Tables XXII to XLIX, collectively, optionally with a proviso that it is not an entire protein of FIG. 2;

(VI) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII-XLIX, optionally with a proviso that it is not an entire protein of FIG. 2;

(VII) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII to XLIX collectively, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;

(VIII) a protein that comprises at least one peptide selected from the peptides set forth in Tables VIII-XXI; and at least one peptide selected from the peptides set forth in Tables XXII to XLIX, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;

(IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A-3R in any whole number increment up to 328, 236, 181, 178, 1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;

(X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A-3R in any whole number increment up to 328, 236, 181, 178, 1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;

(XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A-3R, in any whole number increment up to 328, 236, 181, 178, 1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;

(XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A-3R, in any whole number increment up to 328, 236, 181, 178, 1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;

(XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3A-3R in any whole number increment up to 328, 236, 181, 178, 1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;

(XIV) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively;

(XV) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively;

(XVI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively;

(XVII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX, collectively;

(XVIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX;

(XIX) a peptide that occurs at least once in Tables VIII-XXI, and at least twice in tables XXII to XLIX;

(XX) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX;

(XXI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX;

(XXII) a peptide which comprises one two, three, four, or five of the following characteristics, or an oligonucleotide encoding such peptide:

i) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of FIG. 5;

ii) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of FIG. 6;

iii) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of FIG. 7;

iv) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of FIG. 8; or,

v) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of FIG. 9;

(XXIII) a composition comprising a peptide of (I)-(XXII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form.

(XXIV) a method of using a peptide of (I)-(XXII), or an antibody or binding region thereof or a composition of (XXIII) in a method to modulate a cell expressing 158P3D2;

(XXV) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 158P3D2;

(XXVI) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 158P3D2, said cell from a cancer of a tissue listed in Table I;

(XXVII) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat a cancer;

(XXVIII) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat a cancer of a tissue listed in Table I;

(XXIX) a method of using a a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition and;

(XXIII) in a method to identify or characterize a modulator of a cell expressing 158P3D2.

As used herein, a range is understood to specifically disclose all whole unit positions thereof.

Typical embodiments of the invention disclosed herein include 158P3D2 polynucleotides that encode specific portions of 158P3D2 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145; 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, and 328 or more contiguous amino acids of 158P3D2 variant 1; the maximal lengths relevant for other variants are shown in FIGS. 2A-2Q and 3A-3R.

In general, naturally occurring allelic variants of human 158P3D2 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 158P3D2 protein contain conservative amino acid substitutions within the 158P3D2 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 158P3D2. One class of 158P3D2 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 158P3D2 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 158P3D2 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 158P3D2 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 158P3D2 variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 158P3D2 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 158P3D2 protein having an amino acid sequence of FIG. 3. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds to a 158P3D2 variant also specifically binds to a 158P3D2 protein having an amino acid sequence set forth in FIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG. 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 158P3D2 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.

Other classes of 158P3D2-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of FIG. 3, or a fragment thereof. Another specific class of 158P3D2 protein variants or analogs comprises one or more of the 158P3D2 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 158P3D2 fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 158P3D2 protein shown in FIG. 2 or FIG. 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a 158P3D2 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 158P3D2 amino acid sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 158P3D2 protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.

158P3D2-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 158P3D2-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 158P3D2 protein (or variants, homologs or analogs thereof).

III.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed herein include 158P3D2 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 158P3D2 polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites (see, e.g., URL addresses: pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html; psort.ims.u-tokyo.acjp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsit1.html; Epimatrix™ and Epimer™, Brown University, brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, bimas.dcrt.nih.gov/.).

Motif bearing subsequences of all 158P3D2 variant proteins are set forth and identified in Tables VIII-XXI and XXII-XLIX.

Table V sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns of Table V list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.

Polypeptides comprising one or more of the 158P3D2 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 158P3D2 motifs discussed above are associated with growth dysregulation and because 158P3D2 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables VIII-XXI and XXII-XLIX. CTL epitopes can be determined using specific algorithms to identify peptides within a 158P3D2 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University, URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.

Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For example, on the basis of residues defined in Table IV, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue; substitute a less-preferred residue with a preferred residue; or substitute an originally-occurring preferred residue with another preferred residue. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table IV.

A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 97/33602 to Chesnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the invention include polypeptides comprising combinations of the different motifs set forth in Table VI, and/or, one or more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX, and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically, the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.

158P3D2-related proteins are embodied in many forms, preferably in isolated form. A purified 158P3D2 protein molecule will be substantially free of other proteins or molecules that impair the binding of 158P3D2 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 158P3D2-related proteins include purified 158P3D2-related proteins and functional, soluble 158P3D2-related proteins. In one embodiment, a functional, soluble 158P3D2 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

The invention also provides 158P3D2 proteins comprising biologically active fragments of a 158P3D2 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 158P3D2 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 158P3D2 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starting protein.

158P3D2-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-158P3D2 antibodies or T cells or in identifying cellular factors that bind to 158P3D2. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identify peptides within a 158P3D2 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web URL syfpeithi.bmi-heidelberg.com/; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, Brown University, URL (brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 158P3D2 that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 158P3D2 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation or exon junction, and for HLA Class II predictions 14 flanking residues on either side of a point mutation or exon junction corresponding to that variant, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI, at URL syfpeithi.bmi-heidelberg.com/.

The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of 158P3D2 predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI and XXII-XLVII, selected candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVI-XLIX, selected candidates, 15-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37° C. at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.

It is to be appreciated that every epitope predicted by the BIMAS site, Epimer™ and Epimatrix™ sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be “applied” to a 158P3D2 protein in accordance with the invention. As used in this context “applied” means that a 158P3D2 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a 158P3D2 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 158P3D2-Related Proteins

In an embodiment described in the examples that follow, 158P3D2 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 158P3D2 with a C-terminal 6× His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 158P3D2 protein in transfected cells. The secreted HIS-tagged 158P3D2 in the culture media can be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of 158P3D2-Related Proteins

Modifications of 158P3D2-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 158P3D2 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a 158P3D2 protein. Another type of covalent modification of a 158P3D2 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 158P3D2 comprises linking a 158P3D2 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 158P3D2-related proteins of the present invention can also be modified to form a chimeric molecule comprising 158P3D2 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a 158P3D2 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimeric molecule can comprise multiples of the same subsequence of 158P3D2. A chimeric molecule can comprise a fusion of a 158P3D2-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl-terminus of a 158P3D2 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 158P3D2-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 158P3D2 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 158P3D2-Related Proteins

The proteins of the invention have a number of different specific uses. As 158P3D2 is highly expressed in prostate and other cancers, 158P3D2-related proteins are used in methods that assess the status of 158P3D2 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 158P3D2 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 158P3D2-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 158P3D2 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 158P3D2-related proteins that contain the amino acid residues of one or more of the biological motifs in a 158P3D2 protein are used to screen for factors that interact with that region of 158P3D2.

158P3D2 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 158P3D2 protein), for identifying agents or cellular factors that bind to 158P3D2 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.

Proteins encoded by the 158P3D2 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a 158P3D2 gene product. Antibodies raised against a 158P3D2 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 158P3D2 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 158P3D2-related nucleic acids or proteins are also used in generating HTL or CTL responses.

Various immunological assays useful for the detection of 158P3D2 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 158P3D2-expressing cells (e.g., in radioscintigraphic imaging methods). 158P3D2 proteins are also particularly useful in generating cancer vaccines, as further described herein.

IV.) 158P3D2 ANTIBODIES

Another aspect of the invention provides antibodies that bind to 158P3D2-related proteins. Preferred antibodies specifically bind to a 158P3D2-related protein and do not bind (or bind weakly) to peptides or proteins that are not 158P3D2-related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris-buffered saline containing 25 mM Tris and 150 mM NaCl; or normal saline (0.9% NaCl); 4) animal serum such as human serum; or, 5) a combination of any of 1) through 4); these reactions preferably taking place at pH 7.5, alternatively in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4° C. to 37° C. For example, antibodies that bind 158P3D2 can bind 158P3D2-related proteins such as the homologs or analogs thereof.

158P3D2 antibodies of the invention are particularly useful in cancer (see, e.g., Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 158P3D2 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 158P3D2 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for the detection and quantification of 158P3D2 and mutant 158P3D2-related proteins. Such assays can comprise one or more 158P3D2 antibodies capable of recognizing and binding a 158P3D2-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 158P3D2 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 158P3D2 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 158P3D2 expressing cancers such as prostate cancer.

158P3D2 antibodies are also used in methods for purifying a 158P3D2-related protein and for isolating 158P3D2 homologues and related molecules. For example, a method of purifying a 158P3D2-related protein comprises incubating a 158P3D2 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 158P3D2-related protein under conditions that permit the 158P3D2 antibody to bind to the 158P3D2-related protein; washing the solid matrix to eliminate impurities; and eluting the 158P3D2-related protein from the coupled antibody. Other uses of 158P3D2 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 158P3D2 protein.

Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 158P3D2-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of 158P3D2 can also be used, such as a 158P3D2 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 158P3D2-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used (with or without purified 158P3D2-related protein or 158P3D2 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a 158P3D2 protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 158P3D2 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 158P3D2 amino acid sequence are used to identify hydrophilic regions in the 158P3D2 structure. Regions of a 158P3D2 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 158P3D2 antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of a 158P3D2 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.

158P3D2 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 158P3D2-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 158P3D2 protein can also be produced in the context of chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 158P3D2 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 158P3D2 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 158P3D2 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S. Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598 issued 5 Sep. 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.

Reactivity of 158P3D2 antibodies with a 158P3D2-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 158P3D2-related proteins, 158P3D2-expressing cells or extracts thereof. A 158P3D2 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 158P3D2 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

V.) 158P3D2 CELLULAR IMMUNE RESPONSES

The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world-wide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URL (134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity, including:

1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or 51Cr-release assay involving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a 51Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response “naturally”, or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T cells. At the end of the culture period, T cell activity is detected using assays including 51 Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.

VI.) 158P3D2 TRANSGENIC ANIMALS

Nucleic acids that encode a 158P3D2-related protein can also be used to generate either transgenic animals or “knock out” animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 158P3D2 can be used to clone genomic DNA that encodes 158P3D2. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 158P3D2. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat. No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would be targeted for 158P3D2 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 158P3D2 can be used to examine the effect of increased expression of DNA that encodes 158P3D2. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of 158P3D2 can be used to construct a 158P3D2 “knock out” animal that has a defective or altered gene encoding 158P3D2 as a result of homologous recombination between the endogenous gene encoding 158P3D2 and altered genomic DNA encoding 158P3D2 introduced into an embryonic cell of the animal. For example, cDNA that encodes 158P3D2 can be used to clone genomic DNA encoding 158P3D2 in accordance with established techniques. A portion of the genomic DNA encoding 158P3D2 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 158P3D2 polypeptide.

VII.) METHODS FOR THE DETECTION OF 158P3D2

Another aspect of the present invention relates to methods for detecting 158P3D2 polynucleotides and 158P3D2-related proteins, as well as methods for identifying a cell that expresses 158P3D2. The expression profile of 158P3D2 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 158P3D2 gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 158P3D2 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.

More particularly, the invention provides assays for the detection of 158P3D2 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 158P3D2 polynucleotides include, for example, a 158P3D2 gene or fragment thereof, 158P3D2 mRNA, alternative splice variant 158P3D2 mRNAs, and recombinant DNA or RNA molecules that contain a 158P3D2 polynucleotide. A number of methods for amplifying and/or detecting the presence of 158P3D2 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 158P3D2 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a 158P3D2 polynucleotides as sense and antisense primers to amplify 158P3D2 cDNAs therein; and detecting the presence of the amplified 158P3D2 cDNA. Optionally, the sequence of the amplified 158P3D2 cDNA can be determined.

In another embodiment, a method of detecting a 158P3D2 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 158P3D2 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 158P3D2 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 158P3D2 nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of a 158P3D2 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 158P3D2-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 158P3D2-related protein in a biological sample comprises first contacting the sample with a 158P3D2 antibody, a 158P3D2-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 158P3D2 antibody; and then detecting the binding of 158P3D2-related protein in the sample.

Methods for identifying a cell that expresses 158P3D2 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 158P3D2 gene comprises detecting the presence of 158P3D2 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 158P3D2 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 158P3D2, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 158P3D2 gene comprises detecting the presence of 158P3D2-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 158P3D2-related proteins and cells that express 158P3D2-related proteins.

158P3D2 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 158P3D2 gene expression. For example, 158P3D2 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 158P3D2 expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 158P3D2 expression by RT-PCR, nucleic acid hybridization or antibody binding.

VIII.) METHODS FOR MONITORING THE STATUS OF 158P3D2-RELATED GENES AND THEIR PRODUCTS

Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 158P3D2 expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of 158P3D2 in a biological sample of interest can be compared, for example, to the status of 158P3D2 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology). An alteration in the status of 158P3D2 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol. 1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare 158P3D2 status in a sample.

The term “status” in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 158P3D2 expressing cells) as well as the level, and biological activity of expressed gene products (such as 158P3D2 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 158P3D2 comprises a change in the location of 158P3D2 and/or 158P3D2 expressing cells and/or an increase in 158P3D2 mRNA and/or protein expression.

158P3D2 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 158P3D2 gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 158P3D2 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 158P3D2 gene), Northern analysis and/or PCR analysis of 158P3D2 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 158P3D2 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 158P3D2 proteins and/or associations of 158P3D2 proteins with polypeptide binding partners). Detectable 158P3D2 polynucleotides include, for example, a 158P3D2 gene or fragment thereof, 158P3D2 mRNA, alternative splice variants, 158P3D2 mRNAs, and recombinant DNA or RNA molecules containing a 158P3D2 polynucleotide.

The expression profile of 158P3D2 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 158P3D2 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 158P3D2 status and diagnosing cancers that express 158P3D2, such as cancers of the tissues listed in Table I. For example, because 158P3D2 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 158P3D2 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 158P3D2 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

The expression status of 158P3D2 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 158P3D2 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.

As described above, the status of 158P3D2 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 158P3D2 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 158P3D2 expressing cells (e.g. those that express 158P3D2 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 158P3D2-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 158P3D2 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000);Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 158P3D2 gene products by determining the status of 158P3D2 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 158P3D2 gene products in a corresponding normal sample. The presence of aberrant 158P3D2 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.

In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 158P3D2 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 158P3D2 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 158P3D2 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 158P3D2 mRNA or express it at lower levels.

In a related embodiment, 158P3D2 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 158P3D2 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 158P3D2 expressed in a corresponding normal sample. In one embodiment, the presence of 158P3D2 protein is evaluated, for example, using immunohistochemical methods. 158P3D2 antibodies or binding partners capable of detecting 158P3D2 protein expression are used in a variety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 158P3D2 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 158P3D2 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 158P3D2 indicates a potential loss of function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of 158P3D2 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S. Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of a 158P3D2 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.

Gene amplification is an additional method for assessing the status of 158P3D2. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 158P3D2 expression. The presence of RT-PCR amplifiable 158P3D2 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 158P3D2 mRNA or 158P3D2 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 158P3D2 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 158P3D2 in prostate or other tissue is examined, with the presence of 158P3D2 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 158P3D2 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 158P3D2 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 158P3D2 mRNA or 158P3D2 protein expressed by tumor cells, comparing the level so determined to the level of 158P3D2 mRNA or 158P3D2 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 158P3D2 mRNA or 158P3D2 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 158P3D2 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 158P3D2 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 158P3D2 mRNA or 158P3D2 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 158P3D2 mRNA or 158P3D2 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 158P3D2 mRNA or 158P3D2 protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 158P3D2 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 158P3D2 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 158P3D2 gene and 158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 158P3D2 gene and 158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.

In one embodiment, methods for observing a coincidence between the expression of 158P3D2 gene and 158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 gene products) and another factor associated with malignancy entails detecting the overexpression of 158P3D2 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 158P3D2 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 158P3D2 and PSA mRNA in prostate tissue is examined, where the coincidence of 158P3D2 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 158P3D2 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 158P3D2 mRNA include in situ hybridization using labeled 158P3D2 riboprobes, Northern blot and related techniques using 158P3D2 polynucleotide probes, RT-PCR analysis using primers specific for 158P3D2, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 158P3D2 mRNA expression. Any number of primers capable of amplifying 158P3D2 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 158P3D2 protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) IDENTIFICATION OF MOLECULES THAT INTERACT WITH 158P3D2

The 158P3D2 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 158P3D2, as well as pathways activated by 158P3D2 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the “two-hybrid assay”). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523 issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S. Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).

Alternatively one can screen peptide libraries to identify molecules that interact with 158P3D2 protein sequences. In such methods, peptides that bind to 158P3D2 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 158P3D2 protein(s).

Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 158P3D2 protein sequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3 Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 158P3D2 are used to identify protein-protein interactions mediated by 158P3D2. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 158P3D2 protein can be immunoprecipitated from 158P3D2-expressing cell lines using anti-158P3D2 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 158P3D2 and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 35S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 158P3D2 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 158P3D2's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 158P3D2-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 158P3D2 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 158P3D2 function can be identified based on their ability to bind 158P3D2 and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 158P3D2 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators, which activate or inhibit 158P3D2.

An embodiment of this invention comprises a method of screening for a molecule that interacts with a 158P3 D2 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 158P3D2 amino acid sequence, allowing the population of molecules and the 158P3D2 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 158P3D2 amino acid sequence, and then separating molecules that do not interact with the 158P3D2 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the 158P3D2 amino acid sequence. The identified molecule can be used to modulate a function performed by 158P3D2. In a preferred embodiment, the 158P3D2 amino acid sequence is contacted with a library of peptides.

X.) THERAPEUTIC METHODS AND COMPOSITIONS

The identification of 158P3D2 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in cancers such as those listed in Table I, opens a number of therapeutic approaches to the treatment of such cancers.

Of note, targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues, even vital normal organ tissues. A vital organ is one that is necessary to sustain life, such as the heart or colon. A non-vital organ is one that can be removed whereupon the individual is still able to survive. Examples of non-vital organs are ovary, breast, and prostate.

For example, Herceptin® is an FDA approved pharmaceutical that has as its active ingredient an antibody which is immunoreactive with the protein variously known as HER2, HER2/neu, and erb-b-2. It is marketed by Genentech and has been a commercially successful antitumor agent. Herceptin sales reached almost $400 million in 2002. Herceptin is a treatment for HER2 positive metastatic breast cancer. However, the expression of HER2 is not limited to such tumors. The same protein is expressed in a number of normal tissues. In particular, it is known that HER2/neu is present in normal kidney and heart, thus these tissues are present in all human recipients of Herceptin. The presence of HER2/neu in normal kidney is also confirmed by Latif, Z., et al., B.J.U. International (2002) 89:5-9. As shown in this article (which evaluated whether renal cell carcinoma should be a preferred indication for anti-HER2 antibodies such as Herceptin) both protein and mRNA are produced in benign renal tissues. Notably, HER2/neu protein was strongly overexpressed in benign renal tissue.

Despite the fact that HER2/neu is expressed in such vital tissues as heart and kidney, Herceptin is a very useful, FDA approved, and commercially successful drug. The effect of Herceptin on cardiac tissue, i.e., “cardiotoxicity,” has merely been a side effect to treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very low percentage of patients.

Of particular note, although kidney tissue is indicated to exhibit normal expression, possibly even higher expression than cardiac tissue, kidney has no appreciable Herceptin side effect whatsoever. Moreover, of the diverse array of normal tissues in which HER2 is expressed, there is very little occurrence of any side effect. Only cardiac tissue has manifested any appreciable side effect at all. A tissue such as kidney, where HER2/neu expression is especially notable, has not been the basis for any side effect.

Furthermore, favorable therapeutic effects have been found for antitumor therapies that target epidermal growth factor receptor (EGFR). EGFR is also expressed in numerous normal tissues. There have been very limited side effects in normal tissues following use of anti-EGFR therapeutics.

Thus, expression of a target protein in normal tissue, even vital normal tissue, does not defeat the utility of a targeting agent for the protein as a therapeutic for certain tumors in which the protein is also overexpressed.

Accordingly, therapeutic approaches that inhibit the activity of a 158P3D2 protein are useful for patients suffering from a cancer that expresses 158P3D2. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 158P3D2 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 158P3D2 gene or translation of 158P3D2 mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 158P3D2-related protein or 158P3D2-related nucleic acid. In view of the expression of 158P3D2, cancer vaccines prevent and/or treat 158P3D2-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 158P3D2-related protein, or a 158P3D2-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 158P3D2 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med 1999 February 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 June 49(3): 123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 158P3D2 protein shown in FIG. 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, a 158P3D2 immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 158P3D2 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 158P3D2 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.

In patients with 158P3D2-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, etc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.

X.A.1. Cellular Vaccines

CTL epitopes can be determined using specific algorithms to identify peptides within 158P3D2 protein that bind corresponding HLA alleles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University (URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 158P3D2 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables VIII-XXI and XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.

X.A.2. Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. a 158P3D2 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 158P3D2 in a host, by contacting the host with a sufficient amount of at least one 158P3D2 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 158P3D2 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 158P3D2-related protein or a man-made multiepitopic peptide comprising: administering 158P3D2 immunogen (e.g. a 158P3D2 protein or a peptide fragment thereof, a 158P3D2 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such as a PADRE™ peptide (Epimmune Inc., San Diego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 158P3D2 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a 158P3D2 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 158P3D2, in order to generate a response to the target antigen.

X.A.3. Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 158P3D2. Constructs comprising DNA encoding a 158P3D2-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 158P3D2 protein/immunogen. Alternatively, a vaccine comprises a 158P3D2-related protein. Expression of the 158P3D2-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 158P3D2 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 158P3D2-related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.

Thus, gene delivery systems are used to deliver a 158P3D2-related nucleic acid molecule. In one embodiment, the full-length human 158P3D2 cDNA is employed. In another embodiment, 158P3D2 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.

X.A.4. Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DC) to present 158P3D2 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 158P3D2 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 158P3D2 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 158P3D2 protein. Yet another embodiment involves engineering the overexpression of a 158P3D2 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells that express 158P3D2 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.

X.B.) 158P3D2 as a Target for Antibody-Based Therapy

158P3D2 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because 158P3D2 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 158P3D2-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of 158P3D2 are useful to treat 158P3D2-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

158P3D2 antibodies can be introduced into a patient such that the antibody binds to 158P3D2 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 158P3D2, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a 158P3D2 sequence shown in FIG. 2 or FIG. 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. 158P3D2), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti-158P3D2 antibody) that binds to a marker (e.g. 158P3D2) expressed, accessible to binding or localized on the cell surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 158P3D2, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 158P3D2 epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-158P3D2 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y91 or I131 to anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 158P3D2 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG4 kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

Although 158P3D2 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.

Although 158P3D2 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 158P3D2 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 158P3D2 imaging, or other techniques that reliably indicate the presence and degree of 158P3D2 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.

Anti-158P3D2 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-158P3D2 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-158P3D2 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 158P3D2. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a particular anti-158P3D2 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.

In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 158P3D2 antigen with high affinity but exhibit low or no antigenicity in the patient.

Therapeutic methods of the invention contemplate the administration of single anti-158P3D2 mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti-158P3D2 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-158P3D2 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.

Anti-158P3D2 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-158P3D2 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-158P3D2 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90-minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 158P3D2 expression in the patient, the extent of circulating shed 158P3D2 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.

Optionally, patients should be evaluated for the levels of 158P3D2 in a given sample (e.g. the levels of circulating 158P3D2 antigen and/or 158P3D2 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).

Anti-idiotypic anti-158P3D2 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 158P3D2-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-158P3D2 antibodies that mimic an epitope on a 158P3D2-related protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.

X.C.) 158P3D2 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention. Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly 1-lysine, poly 1-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000)).

Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later development of cells that express or overexpress 158P3D2 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g., in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.

Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, often 200 nM or less; and for Class II an IC50 of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.

4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.

7.) Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.

X.C.1. Minigene Vaccines

A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing epitopes derived 158P3D2, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 158P3D2 (see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.

The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.

Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego, Calif.). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.

Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.

Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.

For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 QYIKANSKFIGITE; (SEQ ID NO: 63), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 DIEKKIAKMEKASSVFNVVNS; (SEQ ID NO: 64), and Streptococcus 18 kD protein at positions 116-131 GAVDSILGGVATYGAA; (SEQ ID NO: 65). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego, Calif.) are designed, most preferably, to bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: xKXVAAWTLKAAx (SEQ ID NO: 66), where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either d-alanine or 1-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include d-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the ε- and α-amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to ε- and α-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to prime specifically an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides

An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 158P3D2. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 158P3D2.

X.D.) Adoptive Immunotherapy

Antigenic 158P3D2-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.

X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 158P3D2. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 158P3D2. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the first diagnosis of 158P3D2-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 158P3D2, a vaccine comprising 158P3D2-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.

It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to stimulate effectively a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.

The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5×109 pfu.

For antibodies, a treatment generally involves repeated administration of the anti-158P3D2 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-158P3D2 mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of 158P3D2 expression in the patient, the extent of circulating shed 158P3D2 antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg body weight followed, e.g., in two, three or four weeks by weekly doses; 0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m2 of body area weekly; 1-600 mg m2 of body area weekly; 225-400 mg m2 of body area weekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 104 cells to about 106 cells, about 106 cells to about 108 cells, about 108 to about 1011 cells, or about 108 to about 5×1010 cells. A dose may also about 106 cells/m2 to about 1010 cells/m2, or about 106 cells/m2 to about 108 cells/m2.

Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) DIAGNOSTIC AND PROGNOSTIC EMBODIMENTS OF 158P3D2

As disclosed herein, 158P3D2 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled “Expression analysis of 158P3D2 in normal tissues, and patient specimens”).

158P3D2 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this disclosure of 158P3D2 polynucleotides and polypeptides (as well as 158P3D2 polynucleotide probes and anti-158P3D2 antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 158P3D2 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays, which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 158P3D2 polynucleotides described herein can be utilized in the same way to detect 158P3D2 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 158P3D2 polypeptides described herein can be utilized to generate antibodies for use in detecting 158P3D2 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 158P3D2 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 158P3D2-expressing cells (lymph node) is found to contain 158P3D2-expressing cells such as the 158P3D2 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

Alternatively 158P3D2 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 158P3D2 or express 158P3D2 at a different level are found to express 158P3D2 or have an increased expression of 158P3D2 (see, e.g., the 158P3D2 expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 158P3D2) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).

The use of immunohistochemistry to identify the presence of a 158P3D2 polypeptide within a tissue section can indicate an altered state of certain cells within that tissue. It is well understood in the art that the ability of an antibody to localize to a polypeptide that is expressed in cancer cells is a way of diagnosing presence of disease, disease stage, progression and/or tumor aggressiveness. Such an antibody can also detect an altered distribution of the polypeptide within the cancer cells, as compared to corresponding non-malignant tissue.

The 158P3D2 polypeptide and immunogenic compositions are also useful in view of the phenomena of altered subcellular protein localization in disease states. Alteration of cells from normal to diseased state causes changes in cellular morphology and is often associated with changes in subcellular protein localization/distribution. For example, cell membrane proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in distribution of the protein in a non-polar manner over the whole cell surface.

The phenomenon of altered subcellular protein localization in a disease state has been demonstrated with MUC1 and Her2 protein expression by use of immunohistochemical means. Normal epithelial cells have a typical apical distribution of MUC1, in addition to some supranuclear localization of the glycoprotein, whereas malignant lesions often demonstrate an apolar staining pattern (Diaz et al, The Breast Journal, 7; 40-45 (2001); Zhang et al, Clinical Cancer Research, 4; 2669-2676 (1998): Cao, et al, The Journal of Histochemistry and Cytochemistry, 45: 1547-1557 (1997)). In addition, normal breast epithelium is either negative for Her2 protein or exhibits only a basolateral distribution whereas malignant cells can express the protein over the whole cell surface (De Potter, et al, International Journal of Cancer, 44; 969-974 (1989): McCormick, et al, 117; 935-943 (2002)). Alternatively, distribution of the protein may be altered from a surface only localization to include diffuse cytoplasmic expression in the diseased state. Such an example can be seen with MUC1 (Diaz, et al, The Breast Journal, 7: 40-45 (2001)).

Alteration in the localization/distribution of a protein in the cell, as detected by immunohistochemical methods, can also provide valuable information concerning the favorability of certain treatment modalities. This last point is illustrated by a situation where a protein may be intracellular in normal tissue, but cell surface in malignant cells; the cell surface location makes the cells favorably amenable to antibody-based diagnostic and treatment regimens. When such an alteration of protein localization occurs for 158P3D2, the 158P3D2 protein and immune responses related thereto are very useful. Accordingly, the ability to determine whether alteration of subcellular protein localization occurred for 24P4C12 make the 158P3D2 protein and immune responses related thereto very useful. Use of the 158P3D2 compositions allows those skilled in the art to make important diagnostic and therapeutic decisions.

Immunohistochemical reagents specific to 158P3D2 are also useful to detect metastases of tumors expressing 158P3D2 when the polypeptide appears in tissues where 158P3D2 is not normally produced.

Thus, 158P3D2 polypeptides and antibodies resulting from immune responses thereto are useful in a variety of important contexts such as diagnostic, prognostic, preventative and/or therapeutic purposes known to those skilled in the art.

Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 158P3D2 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled “Expression analysis of 158P3D2 in normal tissues, and patient specimens,” where a 158P3D2 polynucleotide fragment is used as a probe to show the expression of 158P3D2 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996 Nov.-Dec. 11 (6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a 158P3D2 polynucleotide shown in FIG. 2 or variant thereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. 158P3D2 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 158P3D2 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a 158P3D2 polypeptide shown in FIG. 3).

As shown herein, the 158P3D2 polynucleotides and polypeptides (as well as the 158P3D2 polynucleotide probes and anti-158P3D2 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 158P3D2 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 158P3D2 polynucleotides and polypeptides (as well as the 158P3D2 polynucleotide probes and anti-158P3D2 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 158P3D2 polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 158P3D2 gene maps (see the Example entitled “Chromosomal Mapping of 158P3D2” below). Moreover, in addition to their use in diagnostic assays, the 158P3D2-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).

Additionally, 158P3D2-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 158P3D2. For example, the amino acid or nucleic acid sequence of FIG. 2 or FIG. 3, or fragments of either, can be used to generate an immune response to a 158P3D2 antigen. Antibodies or other molecules that react with 158P3D2 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

XII.) INHIBITION OF 158P3D2 PROTEIN FUNCTION

The invention includes various methods and compositions for inhibiting the binding of 158P3D2 to its binding partner or its association with other protein(s) as well as methods for inhibiting 158P3D2 function.

XII.A.) Inhibition of 158P3D2 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 158P3D2 are introduced into 158P3D2 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-158P3D2 antibody is expressed intracellularly, binds to 158P3D2 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as “intrabodies”, are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to target precisely the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.

In one embodiment, intrabodies are used to capture 158P3D2 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 158P3D2 intrabodies in order to achieve the desired targeting. Such 158P3D2 intrabodies are designed to bind specifically to a particular 158P3D2 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 158P3D2 protein are used to prevent 158P3D2 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 158P3D2 from forming transcription complexes with other factors).

In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII.B.) Inhibition of 158P3D2 with Recombinant Proteins

In another approach, recombinant molecules bind to 158P3D2 and thereby inhibit 158P3D2 function. For example, these recombinant molecules prevent or inhibit 158P3D2 from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a 158P3D2 specific antibody molecule. In a particular embodiment, the 158P3D2 binding domain of a 158P3D2 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 158P3D2 ligand binding domains linked to the Fc portion of a human IgG, such as human IgG1. Such IgG portion can contain, for example, the CH2 and CH3 domains and the hinge region, but not the CH1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 158P3D2, whereby the dimeric fusion protein specifically binds to 158P3D2 and blocks 158P3D2 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

XII.C.) Inhibition of 158P3D2 Transcription or Translation

The present invention also comprises various methods and compositions for inhibiting the transcription of the 158P3D2 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 158P3D2 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 158P3D2 gene comprises contacting the 158P3D2 gene with a 158P3D2 antisense polynucleotide. In another approach, a method of inhibiting 158P3D2 mRNA translation comprises contacting a 158P3D2 mRNA with an antisense polynucleotide. In another approach, a 158P3D2 specific ribozyme is used to cleave a 158P3D2 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 158P3D2 gene, such as 158P3D2 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 158P3D2 gene transcription factor are used to inhibit 158P3D2 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

Other factors that inhibit the transcription of 158P3D2 by interfering with 158P3D2 transcriptional activation are also useful to treat cancers expressing 158P3D2. Similarly, factors that interfere with 158P3D2 processing are useful to treat cancers that express 158P3D2. Cancer treatment methods utilizing such factors are also within the scope of the invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 158P3D2 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 158P3D2 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 158P3D2 antisense polynucleotides, ribozymes, factors capable of interfering with 158P3D2 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 158P3D2 to a binding partner, etc.

In vivo, the effect of a 158P3D2 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application WO98/16628 and U.S. Pat. No. 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.

XIII.) IDENTIFICATION, CHARACTERIZATION AND USE OF MODULATORS OF 158P3D2

XIII.A.) Methods to Identify and Use Modulators

In one embodiment, screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the associated phenotype thereby. In another embodiment, having identified differentially expressed genes important in a particular state; screens are performed to identify modulators that alter expression of individual genes, either increase or decrease. In another embodiment, screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.

In addition, screens are done for genes that are induced in response to a candidate agent. After identifying a modulator (one that suppresses a cancer expression pattern leading to a normal expression pattern, or a modulator of a cancer gene that leads to expression of the gene as in normal tissue) a screen is performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent-treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue, and vice versa. These agent-specific sequences are identified and used by methods described herein for cancer genes or proteins. In particular these sequences and the proteins they encode are used in marking or identifying agent-treated cells. In addition, antibodies are raised against the agent-induced proteins and used to target novel therapeutics to the treated cancer tissue sample.

XIII.B.) Gene Expression-Related Assays

Proteins, nucleic acids, and antibodies of the invention are used in screening assays. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a “gene expression profile,” expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Davis, G F, et al, J Biol Screen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-94, 1996).

The cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins or genes are used in screening assays. That is, the present invention comprises methods for screening for compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This is done on a gene itself or by evaluating the effect of drug candidates on a “gene expression profile” or biological function. In one embodiment, expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring after treatment with a candidate agent, see Zlokamik, supra.

A variety of assays are executed directed to the genes and proteins of the invention. Assays are run on an individual nucleic acid or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. “Modulation” in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue a target value of a 10-fold increase in expression by the test compound is often desired. Modulators that exacerbate the type of gene expression seen in cancer are also useful, e.g., as an upregulated target in further analyses.

The amount of gene expression is monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, a gene product itself is monitored, e.g., through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques also allow for quantification of expression.

XIII.C.) Expression Monitoring to Identify Compounds that Modify Gene Expression

In one embodiment, gene expression monitoring, i.e., an expression profile, is monitored simultaneously for a number of entities. Such profiles will typically involve one or more of the genes of FIG. 2. In this embodiment, e.g., cancer nucleic acid probes are attached to biochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Thus, a series, e.g., wells of a microtiter plate, can be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.

Expression monitoring is performed to identify compounds that modify the expression of one or more cancer-associated sequences, e.g., a polynucleotide sequence set out in FIG. 2. Generally, a test modulator is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein of the invention, or interfere with the binding of a cancer protein of the invention and an antibody or other binding partner.

In one embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds,” as compounds for screening, or as therapeutics.

In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to a cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.

As noted above, gene expression monitoring is conveniently used to test candidate modulators (e.g., protein, nucleic acid or small molecule). After the candidate agent has been added and the cells allowed to incubate for a period, the sample containing a target sequence to be analyzed is, e.g., added to a biochip.

If required, the target sequence is prepared using known techniques. For example, a sample is treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.

The target sequence can be labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.

As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise “sandwich assays”, which include the use of multiple probes, as is generally outlined in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.

A variety of hybridization conditions are used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allow formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.

The reactions outlined herein can be accomplished in a variety of ways. Components of the reaction can be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which can be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target. The assay data are analyzed to determine the expression levels of individual genes, and changes in expression levels as between states, forming a gene expression profile.

XIII.D.) Biological Activity-Related Assays

The invention provides methods identify or screen for a compound that modulates the activity of a cancer-related gene or protein of the invention. The methods comprise adding a test compound, as defined above, to a cell comprising a cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of the invention. In another embodiment, a library of candidate agents is tested on a plurality of cells.

In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e., cell-cell contacts). In another example, the determinations are made at different stages of the cell cycle process. In this way, compounds that modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to identify critical structural features of the compound.

In one embodiment, a method of modulating (e.g., inhibiting) cancer cell division is provided; the method comprises administration of a cancer modulator. In another embodiment, a method of modulating (e.g., inhibiting) cancer is provided; the method comprises administration of a cancer modulator. In a further embodiment, methods of treating cells or individuals with cancer are provided; the method comprises administration of a cancer modulator.

In one embodiment, a method for modulating the status of a cell that expresses a gene of the invention is provided. As used herein status comprises such art-accepted parameters such as growth, proliferation, survival, function, apoptosis, senescence, location, enzymatic activity, signal transduction, etc. of a cell. In one embodiment, a cancer inhibitor is an antibody as discussed above. In another embodiment, the cancer inhibitor is an antisense molecule. A variety of cell growth, proliferation, and metastasis assays are known to those of skill in the art, as described herein.

XIII.E.) High Throughput Screening to Identify Modulators

The assays to identify suitable modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.

In one embodiment, modulators evaluated in high throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, are used. In this way, libraries of proteins are made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred. Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes, or ligands and receptors.

XIII.F.) Use of Soft Agar Growth and Colony Formation to Identify and Characterize Modulators

Normal cells require a solid substrate to attach and grow. When cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and once again require a solid substrate to attach to and grow. Soft agar growth or colony formation in assays are used to identify modulators of cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' ability to grow suspended in solid or semisolid media, such as agar.

Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994). See also, the methods section of Garkavtsev et al. (1996), supra.

XIII.G.) Evaluation of Contact Inhibition and Growth Density Limitation to Identify and Characterize Modulators

Normal cells typically grow in a flat and organized pattern in cell culture until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, transformed cells grow to a higher saturation density than corresponding normal cells. This is detected morphologically by the formation of a disoriented monolayer of cells or cells in foci. Alternatively, labeling index with (3H)-thymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue assay will reveal proliferation capacity of cells and the ability of modulators to affect same. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a lower density.

In this assay, labeling index with 3H)-thymidine at saturation density is a preferred method of measuring density limitation of growth. Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with (3H)-thymidine is determined by incorporated cpm.

Contact independent growth is used to identify modulators of cancer sequences, which had led to abnormal cellular proliferation and transformation. A modulator reduces or eliminates contact independent growth, and returns the cells to a normal phenotype.

XIII.H.) Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators

Transformed cells have lower serum dependence than their normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This is in part due to release of various growth factors by the transformed cells. The degree of growth factor or serum dependence of transformed host cells can be compared with that of control. For example, growth factor or serum dependence of a cell is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.

XIII.I.) Use of Tumor-Specific Marker Levels to Identify and Characterize Modulators

Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, e.g., Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B et al).

Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkless et al., J. Biol. Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). For example, tumor specific marker levels are monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.

XIII.J.) Invasiveness into Matrigel to Identify and Characterize Modulators

The degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used. Briefly, the level of invasion of host cells is measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 1251 and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.

XIII.K.) Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators

Effects of cancer-associated sequences on cell growth are tested in transgenic or immune-suppressed organisms. Transgenic organisms are prepared in a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., mammals such as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous cancer gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.

To prepare transgenic chimeric animals, e.g., mice, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells some of which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived according to U.S. Pat. No. 6,365,797, issued 2 Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug. 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).

Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, a genetically athymic “nude” mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCID mouse, a thymectornized mouse, or an irradiated mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host. Transplantable tumor cells (typically about 106 cells) injected into isogenic hosts produce invasive tumors in a high proportion of cases, while normal cells of similar origin will not. In hosts which developed invasive tumors, cells expressing cancer-associated sequences are injected subcutaneously or orthotopically. Mice are then separated into groups, including control groups and treated experimental groups) e.g. treated with a modulator). After a suitable length of time, preferably 4-8 weeks, tumor growth is measured (e.g., by volume or by its two largest dimensions, or weight) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.

XIII.L.) In Vitro Assays to Identify and Characterize Modulators

Assays to identify compounds with modulating activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The level of protein is measured using immunoassays such as Western blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e.g., Northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.

Alternatively, a reporter gene system can be devised using a cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or P-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol. 1998: 9:624).

As outlined above, in vitro screens are done on individual genes and gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself is performed.

In one embodiment, screening for modulators of expression of specific gene(s) is performed. Typically, the expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.

XIII.M.) Binding Assays to Identify and Characterize Modulators

In binding assays in accordance with the invention, a purified or isolated gene product of the invention is generally used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the amount and/or location of protein. Alternatively, cells comprising the cancer proteins are used in the assays.

Thus, the methods comprise combining a cancer protein of the invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein of the invention. Preferred embodiments utilize the human cancer protein; animal models of human disease of can also be developed and used. Also, other analogous mammalian proteins also can be used as appreciated by those of skill in the art. Moreover, in some embodiments variant or derivative cancer proteins are used.

Generally, the cancer protein of the invention, or the ligand, is non-diffusibly bound to an insoluble support. The support can, e.g., be one having isolated sample receiving areas (a microtiter plate, an array, etc.). The insoluble supports can be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports can be solid or porous and of any convenient shape.

Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition to the support is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies which do not sterically block either the ligand binding site or activation sequence when attaching the protein to the support, direct binding to “sticky” or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or ligand/binding agent to the support, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.

Once a cancer protein of the invention is bound to the support, and a test compound is added to the assay. Alternatively, the candidate binding agent is bound to the support and the cancer protein of the invention is then added. Binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.

Of particular interest are assays to identify agents that have a low toxicity for human cells. A wide variety of assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.

A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the invention can be done in a number of ways. The test compound can be labeled, and binding determined directly, e.g., by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps can be utilized as appropriate.

In certain embodiments, only one of the components is labeled, e.g., a protein of the invention or ligands labeled. Alternatively, more than one component is labeled with different labels, e.g., 1125, for the proteins and a fluorophor for the compound. Proximity reagents, e.g., quenching or energy transfer reagents are also useful.

XIII.N.) Competitive Binding to Identify and Characterize Modulators

In one embodiment, the binding of the “test compound” is determined by competitive binding assay with a “competitor.” The competitor is a binding moiety that binds to the target molecule (e.g., a cancer protein of the invention). Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the competitive binding between the test compound and the competitor displaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a time sufficient to allow binding. Incubations are performed at a temperature that facilitates optimal activity, typically between four and 40° C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.

In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein. In this embodiment, either component can be labeled. Thus, e.g., if the competitor is labeled, the presence of label in the post-test compound wash solution indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the label on the support indicates displacement.

In an alternative embodiment, the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor indicates that the test compound binds to the cancer protein with higher affinity than the competitor. Thus, if the test compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, indicates that the test compound binds to and thus potentially modulates the cancer protein of the invention.

Accordingly, the competitive binding methods comprise differential screening to identity agents that are capable of modulating the activity of the cancer proteins of the invention. In this embodiment, the methods comprise combining a cancer protein and a competitor in a first sample. A second sample comprises a test compound, the cancer protein, and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the cancer protein.

Alternatively, differential screening is used to identify drug candidates that bind to the native cancer protein, but cannot bind to modified cancer proteins. For example the structure of the cancer protein is modeled and used in rational drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins. Moreover, such drug candidates that affect the activity of a native cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of such proteins.

Positive controls and negative controls can be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to allow for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples can be counted in a scintillation counter to determine the amount of bound compound.

A variety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides for the requisite binding.

XIII.O.) Use of Polynucleotides to Down-Regulate or Inhibit a Protein of the Invention.

Polynucleotide modulators of cancer can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand-binding molecule, as described in WO 91/04753. Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.

XIII.P.) Inhibitory and Antisense Nucleotides

In certain embodiments, the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.

In the context of this invention, antisense polynucleotides can comprise naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprised by this invention so long as they function effectively to hybridize with nucleotides of the invention. See, e.g., Isis Pharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.

Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.

Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 12 nucleotides, preferably from about 12 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, e.g., Stein &Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).

XIII.Q.) Ribozymes

In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer-associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25: 289-317 (1994) for a general review of the properties of different ribozymes).

The general features of hairpin ribozymes are described, e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are well known to those of skill in the art (see, e.g., WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad. Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).

XIII.R.) Use of Modulators in Phenotypic Screening

In one embodiment, a test compound is administered to a population of cancer cells, which have an associated cancer expression profile. By “administration” or “contacting” herein is meant that the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a peptide) is put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019. Regulatable gene therapy systems can also be used. Once the modulator has been administered to the cells, the cells are washed if desired and are allowed to incubate under preferably physiological conditions for some period. The cells are then harvested and a new gene expression profile is generated. Thus, e.g., cancer tissue is screened for agents that modulate, e.g., induce or suppress, the cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, altering a biological function or a signaling pathway is indicative of modulator activity. By defining such a signature for the cancer phenotype, screens for new drugs that alter the phenotype are devised. With this approach, the drug target need not be known and need not be represented in the original gene/protein expression screening platform, nor does the level of transcript for the target protein need to change. The modulator inhibiting function will serve as a surrogate marker.

As outlined above, screens are done to assess genes or gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.

XIII.S.) Use of Modulators to Affect Peptides of the Invention

Measurements of cancer polypeptide activity, or of the cancer phenotype are performed using a variety of assays. For example, the effects of modulators upon the function of a cancer polypeptide(s) are measured by examining parameters described above. A physiological change that affects activity is used to assess the influence of a test compound on the polypeptides of this invention. When the functional outcomes are determined using intact cells or animals, a variety of effects can be assesses such as, in the case of a cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., by Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGNIP.

XIII.T.) Methods of Identifying Characterizing Cancer-Associated Sequences

Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one embodiment, the invention provides methods for identifying cells containing variant cancer genes, e.g., determining the presence of, all or part, the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques. The invention comprises methods of identifying the cancer genotype of an individual, e.g., determining all or part of the sequence of at least one gene of the invention in the individual. This is generally done in at least one tissue of the individual, e.g., a tissue set forth in Table I, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known cancer gene, i.e., a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This is done using any number of known homology programs, such as BLAST, Bestfit, etc. The presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.

In a preferred embodiment, the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome. The cancer genes are used as probes to determine the chromosomal localization of the cancer genes. Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the cancer gene locus.

XIV.) KITS/ARTICLES OF MANUFACTURE

For use in the laboratory, prognostic, prophylactic, diagnostic and therapeutic applications described herein, kits are within the scope of the invention. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, along with a label or insert comprising instructions for use, such as a use described herein. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a protein or a gene or message of the invention, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence. Kits can comprise a container comprising a reporter, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label; such a reporter can be used with, e.g., a nucleic acid or antibody. The kit can include all or part of the amino acid sequences in FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecule that encodes such amino acid sequences.

The kit of the invention will typically comprise the container described above and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.

A label can be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit. The label can be on or associated with the container. A label a can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table I.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacture containing compositions, such as amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), e.g., materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth in Table I is provided. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass, metal or plastic. The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), cell population(s) and/or antibody(s). In one embodiment, the container holds a polynucleotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose. In another embodiment a container comprises an antibody, binding fragment thereof or specific binding protein for use in evaluating protein expression of 158P3D2 in cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or directions for such uses can be included on or with such container, as can reagents and other compositions or tools used for these purposes. In another embodiment, a container comprises materials for eliciting a cellular or humoral immune response, together with associated indications and/or directions. In another embodiment, a container comprises materials for adoptive immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), together with associated indications and/or directions; reagents and other compositions or tools used for such purpose can also be included.

The container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 158P3D2 and modulating the function of 158P3D2.

The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.

EXAMPLES

Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which is intended to limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 158P3D2 Gene

To isolate genes that are over-expressed in bladder cancer we used the Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from bladder cancer tissues, including invasive transitional cell carcinoma. The 158P3D2 SSH cDNA sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. Included in the driver were also cDNAs derived from 9 other normal tissues. The 158P3D2 cDNA was identified as highly expressed in the bladder cancer tissue pool, with lower expression seen in a restricted set of normal tissues.

The SSH DNA sequence of 312 bp (FIG. 1) shows identity to the fer-1-like 4 (C. elegans) (FER1L4) mRNA. A 158P3D2 cDNA clone 158P3D2-BCP1 of 1994 bp was isolated from bladder cancer cDNA, revealing an ORF of 328 amino acids (FIG. 2, FIG. 3).

Amino acid sequence analysis of 158P3D2 reveals 100% identity over 328 amino acid region to dJ477O4.1.1, a novel protein similar to otoferlin and dysferlin, isoform 1 protein (GenBank Accession CAB89410.1).

The 158P3D2 protein has a transmembrane domain of 23 residues between amino acids 292-313 predicted by the SOSUI Signal program.

Materials and Methods

Human Tissues:

The patient cancer and normal tissues were purchased from different sources such as the NDRI (Philadelphia, Pa.). mRNA for some of the normal tissues were purchased from Clontech, Palo Alto, Calif.

RNA Isolation:

Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): (SEQ ID NO: 67) 5′TTTTGATCAAGCTT₃₀3′ Adaptor 1: (SEQ ID NO: 68) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO: 69) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 70) 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 71) 3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO: 72) 5′CTAATACGACTCACTATAGGGC3′ Nested primer (NP)1: (SEQ ID NO: 73) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nested primer (NP)2: (SEQ ID NO: 74) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in bladder cancer. The SSH reaction utilized cDNA from bladder cancer and normal tissues.

The gene 158P3D2 sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. The SSH DNA sequence (FIG. 1) was identified.

The cDNA derived from of pool of normal bladder tissues was used as the source of the “driver” cDNA, while the cDNA from a pool of bladder cancer tissues was used as the source of the “tester” cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg of poly(A)⁺ RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from the relevant tissue source (see above) with a mix of digested cDNAs derived from the nine normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine, and heart.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:

To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and 0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5 min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 μl from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 μM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10 sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT) 12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used which included an incubation for 50 min at 42° C. with reverse transcriptase followed by RNAse H treatment at 37° C. for 20 min. After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO: 75) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 76) to amplify β-actin. First strand cDNA (5 μl) were amplified in a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 b.p. β-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal β-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 158P3D2 gene, 5 μl of normalized first strand cDNA were analyzed by PCR using 26, and 30 cycles of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities. The primers used for RT-PCR were designed using the 158P3D2 SSH sequence and are listed below:

158P3D2.1 5′ CATCTATGTGAAGAGCTGGGTGAA 3′ (SEQ ID NO: 77) 158P3D2.2 5′ AGGTAGTCAAAGCGGAACACAAAG 3′ (SEQ ID NO: 78)

Additional primers were also designed to test for expression of the different splice variants and these are listed below:

Primer Name Sequence 158P3D2 ex. 17-R GTCCTCCCAGCAACTCCACACA (SEQ ID NO: 79) 158P3D2 ex. 26-F TGTCCCTTCCACCCAACGTGTGC (SEQ ID NO: 80) 158P3D2 ex. 28-R TCCTCCATCTCTCCTTCCTCCTCAG (SEQ ID NO: 81) 158P3D2 ex. 9-F CAGAAACTGGTGGGAGTCAACA (SEQ ID NO: 82) 158P3D2 ex.1-F ATGGCTCTGACGGTAAGCGTGC (SEQ ID NO: 83) 158P3D2 ex.10-F ATAGGCACCTTCAGGATGGACC (SEQ ID NO: 84) 158P3D2 ex.10-R TCCATCCTGAAGGTGCCTATCC (SEQ ID NO: 85) 158P3D2 ex.16-F CAGAGGAGGAGAAAGAGGAGG (SEQ ID NO: 86) 158P3D2 ex.16-R TCCTCTTTCTCCTCCTCTGG (SEQ ID NO: 87) 158P3D2 ex.21-F AGATCCAGAGTCTAATGCTCACG (SEQ ID NO: 88) 158P3D2 ex.21-R CGTGAGCATTAGACTCTGGATC (SEQ ID NO: 89) 158P3D2 ex.27-F AAGGTGTGGAGTCTGAGGTC (SEQ ID NO: 90) 158P3D2 ex.27-R ACCTCAGACTCCACACCTTGC (SEQ ID NO: 91) 158P3D2 ex.34-R ACTCTGACCAGGAGCTTGATG (SEQ ID NO: 92) 158P3D2 ex.40-F ACACGGAGGATGTGGTTCTGG (SEQ ID NO: 93) 158P3D2 ex.43-F TTGAGCTGCTGACTGTGGAGGAG (SEQ ID NO: 94) 158P3D2 ex.43-R TCCTCCACAGTCAGCAGCTC (SEQ ID NO: 95) 158P3D2 ex.44-R TGAGTGTCCAAGGTCAGCGAG (SEQ ID NO: 96) 158P3D2 ex.7-F AGAGAATGAGCTGGAGCTTGAGC (SEQ ID NO: 97) 158P3D2 ex.7-R TCAAGCTCCAGCTCATTCTCTTC (SEQ ID NO: 98) AGS-25 long RT PCR-3′ TAACACCAGAAAGTTCCACGTCAG (SEQ ID NO: 99) AGS-25 long RT PCR-5′ TGACGGTCGCCGTATTTGATC (SEQ ID NO: 100) AGS-25 short RT PCR-3′ GATTGGCTGCCGAGGCTTGA (SEQ ID NO: 101) AGS-25 short RT PCR-5′ TGACGGTCGCCGTATTTGATC (SEQ ID NO: 102)

A typical RT-PCR expression analysis is shown in FIG. 14. RT-PCR expression analysis was performed on first strand cDNAs generated using pools of tissues from multiple samples. The cDNAs were shown to be normalized using beta-actin PCR. Results show strong expression of 158P3D2 in bladder cancer pool, kidney cancer pool and cancer metastasis pool. Expression of 158P3D2 is also detected in colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pool and prostate metastases to lymph node, and vital pool 2, but not vital pool 1.

Example 2 Full Length Cloning of 158P3D2

The 158P3D2 SSH cDNA sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. The SSH cDNA sequence (FIG. 1) was designated 158P3D2. The full-length cDNA clone 158P3D2 v.1 clone 158P3D2-BCP1 and 158P3D2-BCP2 (FIG. 2) were cloned from bladder cancer pool cDNA.

Additional 158P3D2 splice and SNP variants have been identified and these are listed in FIG. 2 and FIG. 3.

Example 3 Chromosomal Mapping of 158P3D2

Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Ala.), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, N.J.), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Md.).

158P3D2 maps to chromosome 8, using 158P3D2 sequence and the NCBI BLAST tool located on the World Wide Web at: (ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).

Example 4 Expression Analysis of 158P3D2 in Normal Tissues and Patient Specimens

Expression analysis by RT-PCR demonstrated that 158P3D2 is strongly expressed in multiple cancer patient specimens, but unrestricted normal tissues (FIG. 14). First strand cDNA was prepared from a panel of 13 normal tissues (brain, heart, kidney, liver, lung, spleen, skeletal muscle, testis, pancreas, colon, stomach) and pools of 4-7 patients from the following cancer indications: bladder, kidney, colon, lung, pancreas, stomach, ovary, breast, multiple cancer metastasis, cervix, lymphoma as well as from a pool of patient-derived xenografts (prostate cancer, bladder cancer and kidney cancer). Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the AlphaImager software. Results show strong expression of 158P3D2 in cancers of the bladder, kidney, colon, lung, pancreas, stomach, ovary, breast, cervix, and lymphoma. Strong expression was also observed in the cancer metastasis pool. Low expression was detected in all normal tissues tested except in normal stomach.

Expression of 158P3D2 in bladder cancer patient specimens and human normal tissues is shown in FIG. 15. First strand cDNA was prepared from normal bladder, bladder cancer cell lines (UM-UC-3, TCCSUP, J82) and a panel of bladder cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in the majority of bladder cancer patient specimens tested. Very low expression was detected in normal tissues, but no expression was seen in the cell lines tested.

Northern blot analysis of 158P3D2 in bladder specimens is shown in FIG. 16. RNA was extracted from normal bladder, bladder cancer cell lines (UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) and their normal adjacent tissues (NAT). Northern blot with 10 μg of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor tissues, but not in normal, nor NAT tissues.

FIG. 17 shows 158P3D2 expression in lung cancer patient specimens. First strand cDNA was prepared from normal lung, cancer cell line A427 and a panel of lung cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in 35/39 (90%) of lung cancer specimens, but not in all 3 normal lung tissues tested.

Northern blot analysis of 158P3D2 expression in lung cancer patient specimens is shown in FIG. 18. RNA was extracted from normal lung, A427 lung cancer cell line, and a panel of lung cancer patient specimens. Northern blot with 10 μg of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in normal tissues.

FIG. 19 shows 158P3D2 expression in cancer metastasis patient specimens. First strand cDNA was prepared from normal colon, kidney, liver, lung, pancreas, stomach and from a panel of cancer metastasis patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in the majority of patient cancer metastasis specimens tested but not in normal tissues.

FIG. 20 shows 158P3D2 expression in cervical cancer patient specimens. First strand cDNA was prepared from normal cervix, cervical cancer cell line HeLa, and a panel of cervical cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show expression of 158P3D2 in all 14 cervical cancer patient specimens tested. No expression was detected in normal cervix or in the cell line tested.

Northern blot analysis of 158P3D2 expression in cervical cancer patient specimens is shown in FIG. 21. RNA was extracted from normal cervix, cervical cancer cell line HeLa, and a panel of cervical cancer patient specimens. Northern blot with 10 μg of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor tissues, but not in normal cervix nor in the cell line.

FIG. 22 shows 158P3D2 expression in kidney cancer patient specimens. First strand cDNA was prepared from normal kidney, kidney cancer cell lines (769-P, A-498, CAKI-1), and a panel of kidney cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of kidney cancer patient specimens, but not in all 3 normal kidney tissues tested. Low expression was detected in 2 of 3 cell lines tested.

FIG. 23 shows 158P3D2 expression in kidney cancer patient specimens by northern blotting. RNA was extracted from normal kidney and a panel of kidney cancer patient specimens. Northern blot with 10 μg of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in the normal tissue.

FIG. 24 shows 158P3D2 expression in stomach cancer patient specimens. First strand cDNA was prepared from normal stomach, and a panel of stomach cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of stomach cancer patient specimens. Weak expression was detected in the 2 normal stomach, and only in 1 of the 2 NAT tissues tested.

FIG. 25 shows 158P3D2 expression in stomach cancer patient specimens by northern blotting. RNA was extracted from normal stomach and a panel of stomach cancer patient specimens. Northern blot with 10 μg of total RNA were probed with the 158P3D2 sequence. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor specimens but not in the normal tissue.

FIG. 26 shows 158P3D2 expression in colon cancer patient specimens. First strand cDNA was prepared from normal colon, colon cancer cell lines (LoVo, CaCO-2, SK CO 1, Colo 205, T284), and a panel of colon cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×s), medium (signal detected at 26×), high (strong signal at 26×). 158P3D2 is expressed at varying levels in the majority of colon cancer patient specimens. But it was weakly expressed in just 2 of 3 normal tissues, and 3 of 5 cell lines tested.

FIG. 27 shows 158P3D2 expression in uterus cancer patient specimens. First strand cDNA was prepared from normal uterus and a panel of uterus cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show 158P3D2 is expressed at varying levels in the majority of uterus cancer patient specimens, but not in normal uterus.

FIG. 28 shows 158P3D2 expression in breast cancer patient specimens. First strand cDNA was prepared from normal breast, breast cancer cell lines (MD-MBA-435S, DU4475, MCF-7, CAMA-1, MCF10A), and a panel of breast cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Expression level was recorded as no expression (no signal detected), low (signal detected at 30×), medium (signal detected at 26×), high (strong signal at 26×). Results show 158P3D2 is expressed at varying levels in the majority of breast cancer patient specimens. But it was weakly expressed in just 2 of 3 normal tissues, and 2 of 5 cell lines tested.

The restricted expression of 158P3D2 in normal tissues and the expression detected in bladder cancer, kidney cancer, colon cancer, lung cancer, pancreas cancer, stomach cancer, ovary cancer, breast cancer, uterus cancer, cervical cancer and lymphoma suggest that 158P3D2 is a potential therapeutic target and a diagnostic marker for the treatment of human cancers.

Example 5 Transcript Variants of 158P3D2

Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding (5′ or 3′ end) portions, from the original transcript. Transcript variants can code for similar or different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in the same tissue at the same time, or in different tissues at the same time, or in the same tissue at different times, or in different tissues at different times. Proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, e.g., secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by full-length cloning experiment, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.

Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” Genome Research. 2000 April; 10(4):516-22); Grail (URL compbio.oml.gov/Grail-bin/EmptyGrailForm) and GenScan (URL genes.mit.edu/GENSCAN.html). For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S. J., et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7; 97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5′ RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6; Ferranti P, et al., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein, Eur J. Biochem. 1997 Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al., Discovery of new human beta-defensins using a genomics-based approach, Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACE Validation: Brigle, K. E., et al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 158P3D2 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 158P3D2 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.

Using the full-length gene and EST sequences, six transcript variants were identified, designated as 158P3D2 v.2, v.14 through v.18. The boundaries of the exon in the original transcript, 158P3D2 v.1 were shown in Table LI. Exon compositions of the variants are shown in FIG. 10. Each different combination of exons in spatial order, e.g. exon 1 of v.2 and exons 3, 4, 5 and 6 of v.1, is a potential splice variant.

Tables LII(a)-(f) through LV(a)-(f) are set forth on a variant-by-variant bases. Tables LII(a)-(f) show nucleotide sequence of the transcript variants. Tables LIII(a)-(f) show the alignment of the respective transcript variant with nucleic acid sequence of 158P3D2 v.1. Tables LIV(a)-(f) lay out amino acid translation of the transcript variants for the identified reading frame orientation. Tables LV(a)-(f) displays alignments of the amino acid sequence encoded by the splice variant with that of 158P3D2 v.1.

Example 6 Single Nucleotide Polymorphisms of 158P3D2

A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNP that occurs on a cDNA is called cSNP. This cSNP may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNP cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNP and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotide polymorphisms in the isolation of common disease genes,” Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. 2000 February; 1(1):15-26).

SNP are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 October-November; 20(9): 18-20; K. M. Weiss, “In search of human variation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. 2001 February; 47(2): 164-172). For example, SNP can be identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNP by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNP can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol. Diagn. 2000 December; 5(4):329-340).

Using the methods described above, twelve SNP were identified in the original transcript, 158P3D2 v.1, at positions 1155 (T/C), 1152 (G/A), 960 (G/T) and 1236 (G/-), 519 (A/G), 440 (T/A), 971 (T/C), 150 (C/G), 1022 (C/A), 1148 (G/A), 1691 (G/T) and 1692 (A/G). The transcripts or proteins with alternative allele were designated as variant 158P3D2 v.3 through v.13, respectively. FIG. 12 shows the schematic alignment of the SNP variants. FIG. 11 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as v.1 are not shown in FIG. 11. These alleles of the SNP, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 158P3D2 v.17) that contains the site of the SNP.

Example 7 Production of Recombinant 158P3D2 in Prokaryotic Systems

To express recombinant 158P3D2 and 158P3D2 variants in prokaryotic cells, the full or partial length 158P3D2 and 158P3D2 variant cDNA sequences are cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 158P3D2 variants are expressed: the full length sequence presented in FIGS. 2 and 3, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs

pCRII: To generate 158P3D2 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) are generated encoding either all or fragments of the 158P3D2 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 158P3D2 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 158P3D2 at the RNA level. Transcribed 158P3D2 RNA representing the cDNA amino acid coding region of the 158P3D2 gene is used during in vitro translation systems such as the TnT™ Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 158P3D2 protein.

B. Bacterial Constructs

pGEX Constructs: To generate recombinant 158P3D2 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pGEX family of GST-fusion vectors (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 158P3D2 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6× His) at the carboxyl-terminus. The GST and 6× His tags permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies. The 6× His tag is generated by adding 6 histidine codons to the cloning primer at the 3′ end, e.g., of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission™ recognition site in pGEX-6P-1, may be employed such that it permits cleavage of the GST tag from 158P3D2-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 158P3D2 proteins that are fused to maltose-binding protein (MBP), all or parts of the 158P3D2 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, Mass.). These constructs allow controlled expression of recombinant 158P3D2 protein sequences with MBP fused at the amino-terminus and a 6× His epitope tag at the carboxyl-terminus. The MBP and 6× His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies. The 6× His epitope tag is generated by adding 6 histidine codons to the 3′ cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 158P3D2. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.

pET Constructs: To express 158P3D2 in bacterial cells, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 158P3D2 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6× His and S-Tag™ that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 158P3D2 protein are expressed as amino-terminal fusions to NusA. The cDNA encoding amino acids 155-290 and amino acids 260-328 of 158P3D2 each were cloned into the pET-21b vector. The recombinant proteins can be used to generate rabbit polyclonal antibodies.

C. Yeast Constructs:

pESC Constructs: To express 158P3D2 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag™ or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 158P3D2. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells.

pESP Constructs: To express 158P3D2 in the yeast species Saccharomyces pombe, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 158P3D2 protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A Flag™ epitope tag allows detection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant 158P3D2 in Higher Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 158P3D2 in eukaryotic cells, the full or partial length 158P3D2 cDNA sequences, or variants thereof, can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 158P3D2 are expressed in these constructs, amino acids 1 to 328, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2 v.1, v.3, v.4, v.10, v.12 and v.13; amino acids 1 to 236, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2v.2A; amino acids 1 to 181, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2 v.2B or v.5B; amino acids 1 to 178, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2 v.5A; amino acids 1 to 2036, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2 v.17; amino acids 1 to 1990, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2v.16; amino acids 1 to 1145, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2 v.15; amino acids 1 to 1393, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2 v.14; amino acids 1 to 610, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2v.18; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 282P1G3 variants, or analogs thereof.

The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-158P3D2 polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 158P3D2 in mammalian cells, a 158P3D2 ORF, or portions thereof, of 158P3D2 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has Xpress™ and six histidine (6× His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs: To express 158P3D2 in mammalian cells, a 158P3D2 ORF, or portions thereof, of 158P3D2 with a consensus Kozak translation initiation site was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6× His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. FIG. 19 shows expression of 158P3D2.pcDNA3.1/mychis in transiently transfected 293T cells.

pcDNA3.1/CT-GFP-TOPO Construct: To express 158P3D2 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 158P3D2 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 158P3D2 protein.

PAPtag: A 158P3D2 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a 158P3D2 protein while fusing the IgGκ signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgGκ signal sequence is fused to the amino-terminus of a 158P3D2 protein. The resulting recombinant 158P3D2 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 158P3D2 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6× His epitopes fused at the carboxyl-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.

ptag5: A 158P3D2 ORF, or portions thereof, is cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 158P3D2 protein with an amino-terminal IgGκ signal sequence and myc and 6× His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 158P3D2 protein is optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 158P3D2 proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

PsecFc: A 158P3D2 ORF, or portions thereof, is also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgG 1 Fc fusion at the carboxyl-terminus of the 158P3D2 proteins, while fusing the IgGK signal sequence to N-terminus. 158P3D2 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 158P3D2 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with 158P3D2 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

pSRα Constructs: To generate mammalian cell lines that express 158P3D2 constitutively, 158P3D2 ORF, or portions thereof, of 158P3D2 were cloned into pSRα constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRα constructs into the 293T-10A 1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 158P3D2, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as the FLAG™ tag to the carboxyl-terminus of 158P3D2 sequences to allow detection using anti-Flag antibodies. For example, the FLAG™ sequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 103) is added to cloning primer at the 3′ end of the ORF. Additional pSRα constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6× His fusion proteins of the full-length 158P3D2 proteins.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 158P3D2. High virus titer leading to high level expression of 158P3D2 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 158P3D2 coding sequences or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 158P3D2 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 158P3D2 in mammalian cells, coding sequences of 158P3D2, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 158P3D2. These vectors are thereafter used to control expression of 158P3D2 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 158P3D2 proteins in a baculovirus expression system, 158P3D2 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-158P3D2 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.

Recombinant 158P3D2 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 158P3D2 protein can be detected using anti-158P3D2 or anti-His-tag antibody. 158P3D2 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 158P3D2 which are used for diagnostic and therapeutic purposes.

Example 9 Antigenicity Profiles and Secondary Structure

FIG. 5A-I, FIG. 6A-I, FIG. 7A-I, FIG. 8A-I, and FIG. 9A-I depict graphically five amino acid profiles of 158P3D2 variants 1, 2a, 2b, 5a, 14, 15, 16, 17, 18, (A) through (I) respectively, each assessment available by accessing the ProtScale website on the ExPasy molecular biology server.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG. 8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of each of the 158P3D2 variant proteins. Each of the above amino acid profiles of 158P3D2 variants were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and Percentage Accessible Residues (FIG. 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determine stretches of amino acids (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.

Antigenic sequences of the 158P3D2 variant proteins indicated, e.g., by the profiles set forth in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and/or FIG. 9 are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-158P3D2 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 158P3D2 protein variants listed in FIGS. 2 and 3. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profiles of FIG. 5; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profiles of FIG. 7; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles on FIG. 8; and, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excipient compatible with human physiology.

The secondary structures of 158P3D2 protein variants 1, 2a, 2b, 5a, 14, 15, 16, 17, and 18, namely the predicted presence and location of alpha helices, extended strands, and random coils, are predicted from their primary amino acid sequences using the HNN—Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deléage G., accessed from the ExPasy molecular biology server. The analysis indicates that 158P3D2 variant 1 is composed of 32.93% alpha helix, 18.29% extended strand, and 48.78% random coil (FIG. 13A). 158P3D2 variant 2a is composed of 25.58% alpha helix, 18.22% extended strand, and 55.93% random coil (FIG. 13B). 158P3D2 variant 2b is composed of 44.75% alpha helix, 11.60% extended strand, and 43.65% random coil (FIG. 13C). 158P3D2 variant 5a is composed of 9.55% alpha helix, 26.40% extended strand, and 64.04% random coil (FIG. 13D). 158P3D2 variant 14 is composed of 33.88% alpha helix, 13.42% extended strand, and 52.69% random coil (FIG. 13E). 158P3D2 variant 15 is composed of 33.28% alpha helix, 15.11% extended strand, and 51.62% random coil (FIG. 13F). 158P3D2 variant 16 is composed of 32.76% alpha helix, 14.47% extended strand, and 52.76% random coil (FIG. 13G). 158P3D2 variant 17 is composed of 32.86% alpha helix, 14.69% extended strand, and 52.46% random coil (FIG. 13H). 158P3D2 variant 18 is composed of 27.21% alpha helix, 14.75% extended strand, and 58.03% random coil (FIG. 13I).

Analysis for the potential presence of transmembrane domains in the 158P3D2 variant proteins 1, 2a, 2b, 5a, 14, 15, 16, 17, and 18, was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server. Shown graphically in FIGS. 13L, 13N, 13P, 13R, 13T, 13V, 13X, 13Z are the results of analysis of variants 1, 2a, 2b, 5a, 14, 15, 16, 17, and 18, respectively, using the TMpred program. Shown graphically in FIGS. 13K, 13M, 13O, 13Q, 13S, 13U, 13W, 13Y, 13AA are the results of analysis of variants 1, 2a, 2b, 5a, 14, 15, 16, 17, and 18, respectively using the TMHMM program. Both programs predict the presence of 1 transmembrane domain in variant 1, Both programs predict that variants 2a, 2b, 5a, and 18 lack transmembrane domains and are soluble proteins. The TMpred program predicts that variants 14, 15, 16, and 17 have 2 transmembrane domains of which the more carboxy-terminal transmembrane has a higher probability of existence. The TMHMM program predicts that variants 14 and 15 do not encode transmembrane domains and variants 16 and 17 contain 1 transmembrane domain. Analyses of the variants using other structural prediction programs are summarized in Table VI and Table L.

Example 10 Generation of 158P3D2 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with a full length 158P3D2 protein variant, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled “Antigenicity Profiles and Secondary Structure”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate such regions of 158P3D2 protein variant 1 or other protein variants).

For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 158P3D2 protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described in Example 11 (“Generation of Monoclonal Antibodies”). For example, in 158P3D2 variant 1, such regions include, but are not limited to, amino acids 1-25, amino acids 37-54, amino acids 60-73, amino acids 187-225, and amino acids 235-271. An extracellular epitope peptide encoding amino acids 315 to 328 is also used to generate antibodies that bind to the extracellular region of 158P3D2 protein. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 315-328 of 158P3D2 variant 1 was conjugated to KLH and used to immunize a rabbit. Alternatively the immunizing agent may include all or portions of the 158P3D2 variant proteins, analogs or fusion proteins thereof. For example, the 158P3D2 variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins.

In one embodiment, amino acids 155-290 of 158P3D2 variant 1 were fused to His using recombinant techniques and the pET21b expression vector. In another embodiment, amino acids 260-328 were cloned into the pET21b expression vector. The proteins are then expressed, purified, and used to immunize rabbits. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.

Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 158P3D2 in Prokaryotic Systems” and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled “Production of Recombinant 158P3D2 in Eukaryotic Systems”), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 1-236 of 158P3D2 variant 2a is cloned into the Tag5 mammalian secretion vector, and expressed in 293T cells. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 158P3D2 protein is then used as immunogen.

During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 μg, typically 100-200 μg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such a rabbit serum derived from immunization with the His-fusion of 158P3D2 variant 1 protein, the full-length 158P3D2 variant 1 cDNA was cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 158P3D2 in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-158P3D2 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 158P3D2 protein using the Western blot technique. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 158P3D2-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 158P3D2 are also carried out to test reactivity and specificity.

Anti-serum from rabbits immunized with 158P3D2 variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a GST-158P3D2 variant 1 fusion protein is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-158P3D2 fusion protein covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.

Example 11 Generation of 158P3D2 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 158P3D2 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would disrupt or modulate the biological function of the 158P3D2 variants, for example those that would disrupt the interaction with ligands and binding partners. Immunogens for generation of such mAbs include those designed to encode or contain the entire 158P3D2 protein variant sequence, regions predicted to contain functional motifs, and regions of the 158P3D2 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9, and the Example entitled “Antigenicity Profiles and Secondary Structure”). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells engineered to express high levels of a respective 158P3D2 variant, such as Rat1-158P3D2 variant 1 or 300.19-158P3D2 variant 1 murine Pre-B cells, were used to immunize mice.

To generate mAbs to a 158P3D2 variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 10⁷ 158P3D2-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding a 158P3D2 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 1-400 of 158P3D2 of variant 15 is cloned into the Tag5 mammalian secretion vector and the recombinant vector will then be used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 158P3D2 variant 15 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 158P3D2 variant.

During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytometric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 158P3D2 monoclonal antibodies, a peptide encoding amino acids 315-328 of 158P3D2 variant 1 was coupled to KLH and use to immunize mice. Balb C mice were immunized with 10 μg of the KLH-peptide mixed in adjuvant. Mice were subsequently immunized over several weeks with the KLH-peptide. ELISA using the peptide coupled to a different carrier, ovalbumin, determined the titer of serum from the immunized mice (See FIG. 29). Reactivity and specificity of the serum to full length 158P3D2 variant 1 protein was monitored by flow cytometry and Western blotting using recombinant 158P3D2 variant 1-expressing cells and cells endogenously expressing 158P3D2 variant 1 protein. Mice showing the strongest reactivity were rested and given a final injection of antigen and sacrificed for fusion. The lymph nodes of the sacrificed mice were harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells were analyzed by flow cytometry to identify specific-158P3D2 surface binding MAbs. Supernatants were also screened by ELISA, Western blot, immunoprecipitation, and fluorescent microscopy to identify 158P3D2 specific antibody-producing clones.

In other embodiments, 158P3D2 variant specific MAbs are generated by employing immunogens that encode amino acid sequences unique to each variant or created by unique junctions from alternative splicing of exons. For example, a peptide encoding amino acids 1018-1035 of 158P3D2 variant 15 is coupled to KLH and used to immunize mice. In another example, amino acids 1375-1393 of 158P3D2 variant 14 is coupled to KLH and used to immunize mice. Hybridomas resulting from fusion of the B-cells from the mice are screened on cells expressing the respective 158P3D2 variant protein from which the antigen was derived and cross-screened on cells expressing the other variant proteins to identify variant specific MAbs and MAbs that may recognize more than 1 variant.

The binding affinity of 158P3D2 variant specific monoclonal antibodies was determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 158P3D2 variant monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.

In addition, equilibrium binding analysis of a dilution series of the MAb was also used to determine affinity defined by the dissociation constant (KD). The KD is determined by non-linear regression of the equilibrium binding data of the concentration series. The KD is defined as the concentration at which half-maximal binding of the MAb to the antigen is attained under equilibrium conditions.

Example 12 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM 125I-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC50≧[HLA], the measured IC50 values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC50 of a positive control for inhibition by the IC50 for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation is accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif-bearing peptides (see Table IV).

Example 13 Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/or Motif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequences in the Example entitled “Antigenicity Profiles” and Tables VIII-XXI and XXII-XLIX employ the protein sequence data from the gene product of 158P3D2 set forth in FIGS. 2 and 3, the specific search peptides used to generate the tables are listed in Table VII.

Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 158P3D2 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type: “ΔG”=a1i×a2i×a3i . . . ×ani

where aji is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount ji to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of ji. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Protein sequences from 158P3D2 are scanned utilizing motif identification software, to identify 8-, 9-10- and 11-mer sequences containing the HLA-A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule).

These peptides are then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptides that bind to at least three of the five A2-supertype alleles tested are typically deemed A2-supertype cross-reactive binders. Preferred peptides bind at an affinity equal to or less than 500 nM to three or more HLA-A2 supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 158P3D2 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3-supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent A3-supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of ≦500 nM, often ≦200 nM, are then tested for binding cross-reactivity to the other common A3-supertype alleles (e.g., A*3101, A*3301, and A*6801) to identify those that can bind at least three of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 158P3D2 protein(s) scanned above is also analyzed for the presence of 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with IC50 of ≦500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules (e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of binding to three or more of the five B7-supertype alleles tested are thereby identified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 158P3D2 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.

Example 14 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:

Target Cell Lines for Cellular Screening:

The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or transfectants comprising the gene encoding the antigen of interest, can be used as target cells to confirm the ability of peptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytes are purified by plating 10×106 PBMC/well in a 6-well plate. After 2 hours at 37° C., the non-adherent cells are removed by gently shaking the plates and aspirating the supernatants. The wells are washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the Detacha-Bead® reagent. Typically about 200-250×106 PBMC are processed to obtain 24×106 CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20×106 cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140 μl beads/20×106 cells) and incubated for 1 hour at 4° C. with continuous mixing. The beads and cells are washed 4× with PBS/AB serum to remove the nonadherent cells and resuspended at 100×106 cells/ml (based on the original cell number) in PBS/AB serum containing 100 μl/ml Detacha-Bead® reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with μg/ml of peptide at a cell concentration of 1-2×106/ml in the presence of 3 μg/ml B2-microglobulin for 4 hours at 20° C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×105 cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×106 cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human IL-10 is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10 IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5×106 cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at 2×106 in 0.5 ml complete medium per well and incubated for 2 hours at 37° C. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10 μg/ml of peptide in the presence of 3 μg/ml β2 microglobulin in 0.25 ml RPMI/5% AB per well for 2 hours at 37° C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a 51 Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNγ ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.

Measurement of CTL Lytic Activity by 51Cr Release.

Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 51Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200 μCi of 51Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled target cells are resuspended at 106 per ml and diluted 1:10 with K562 cells at a concentration of 3.3×106/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (10011) are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the formula: [(cpm of the test sample−cpm of the spontaneous 51Cr release sample)/(cpm of the maximal 51Cr release sample−cpm of the spontaneous 51Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample-background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.

In Situ Measurement of Human IFNγ Production as an Indicator of Peptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHCO3, pH8.2) overnight at 4° C. The plates are washed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 l/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of 1×106 cells/ml. The plates are incubated for 48 hours at 37° C. with 5% CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200 μg/100 microliter/well and the plate incubated for two hours at 37° C. The plates are washed and 100 μl of biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3% FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6× with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well 1M H3PO4 and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×105 irradiated (8,000 rad) EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25 μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 200 IU/ml and every three days thereafter with fresh media at 50 IU/ml. The cells are split if the cell concentration exceeds 1×106/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assay or at 1×106/ml in the in situ IFNγ assay using the same targets as before the expansion.

Cultures are expanded in the absence of anti-CD3+ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. and irradiated (4,200 rad); 2×105 irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10% (v/v) human AB serum, non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptide-specific CTLs in at least individuals, and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 158P3D2. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified as set forth herein are confirmed in a manner analogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc. are also confirmed using similar methodology.

Example 15 Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For example, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*0201, then, if A*0201 binding capacity is maintained, for A2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertype members and then analoged to modulate binding affinity to any one (or more) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the capacity of the parent wild type (WT) peptide to bind at least weakly, i.e., bind at an IC50 of 500 nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analoged peptides have been shown to have increased immunogenicity and cross-reactivity by T cells specific for the parent epitope (see, e.g., Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA 92:8166, 1995).

In the cellular screening of these peptide analogs, it is important to confirm that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies similar to those employed in analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to ⅗ of the A3-supertype molecules are engineered at primary anchor residues to possess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 and A*11 (prototype A3 supertype alleles). Those peptides that demonstrate ≦500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. B7 supermotif-bearing peptides are, for example, engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay. Again, it is generally important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of a B7 supermotif-bearing peptide with an F residue at position 1 is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 158P3D2-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substitution of α-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g., the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the binding properties and/or cross-reactivity of peptide ligands for HLA supertype molecules can be modulated.

Example 16 Identification and Confirmation of 158P3D2-Derived Sequences with HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif are identified and confirmed as outlined below using methodology similar to that described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 158P3D2-derived, HLA class II HTL epitopes, a 158P3D2 antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR-supermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have been developed (Southwood et al., J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors (i.e., at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors. Using allele-specific selection tables (see, e.g., Southwood et al., ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.

The 158P3D2-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, and DR9 molecules in secondary assays. Finally, peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, are screened for binding to DR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides binding at least seven of the ten DR molecules comprising the primary, secondary, and tertiary screening assays are considered cross-reactive DR binders. 158P3D2-derived peptides found to bind common HLA-DR alleles are of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epitopes. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 158P3D2 antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 μM or better, i.e., less than 1 μM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides are analoged to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimal residue for DR3 binding, and substitution for that residue often improves DR 3 binding.

Example 17 Immunogenicity of 158P3D2-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous to the determination of immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL responses and/or by using appropriate transgenic mouse models. Immunogenicity is determined by screening for: 1.) in vitro primary induction using normal PBMC or 2.) recall responses from patients who have 158P3D2-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-Supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1−(1−Cgf)2].

Where frequency data is not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies is assumed. To obtain total potential supertype population coverage no linkage disequilibrium is assumed, and only alleles confirmed to belong to each of the supertypes are included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations are made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, A11, A31, A*3301, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%, see, e.g., Table IV (G). An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al., J. Immunol. 159:1648, 1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in identifying candidate epitopes for inclusion in a vaccine that is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from the art), an average population coverage is predicted to be greater than 95% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994), can be used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is 95%.

Example 19 CTL Recognition of Endogenously Processed Antigens after Priming

This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as described herein recognize endogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells in the absence or presence of peptide, and also tested on ⁵¹Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 158P3D2 expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 158P3D2 antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/K^(b) transgenic mice, several other transgenic mouse models including mice with human A11, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.

Example 20 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 158P3D2-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 158P3D2-expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997). For example, A2/K^(b) mice, which are transgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991).

In vitro CTL activation: One week after priming, spleen cells (30×10⁶ cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) are incubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 10⁴ ⁵¹Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a six hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release=100×(experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Cr release assay. To obtain specific lytic units/10⁶, the lytic units/10⁶ obtained in the absence of peptide is subtracted from the lytic units/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Cr release is obtained at the effector (E): target (T) ratio of 50:1 (i.e., 5×10⁵ effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(1/50,000)−(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in the Example entitled “Confirmation of Immunogenicity.” Analyses similar to this may be performed to conform the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in a 158P3D2-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or can be single and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responses that are correlated with 158P3D2 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 158P3D2. For example, if it has been observed that patients who spontaneously clear 158P3D2-expressing cells generate an immune response to at least three (3) epitopes from 158P3D2 antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of 500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site, at URL bimas.dcrt.nih.gov/.

In order to achieve broad coverage of the vaccine through out a diverse population, sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. In one embodiment, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. Epitopes may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. A multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent the creating of any analogs) directs the immune response to multiple peptide sequences that are actually present in 158P3D2, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 158P3D2.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epitope analogs as described herein.

A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 158P3D2, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 158P3D2 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.

This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.

The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM (NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to which it Induces Immunogenicity

The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vitro by determining epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines “antigenicity” and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al., J. Immunol. 154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761, 1994.

For example, to confirm the capacity of a DNA minigene construct containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a ⁵¹Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is also found that the minigene elicits appropriate immune responses directed toward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, I-A^(b)-restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a ³H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.

DNA minigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein (e.g., Barnett et al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med. 5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in the Example entitled “Induction of CTL Responses Using a Prime Boost Protocol.”

Example 24 Peptide Compositions for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent 158P3D2 expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, is administered to individuals at risk for a 158P3D2-associated tumor.

For example, a peptide-based composition is provided as a single polypeptide that encompasses multiple epitopes. The vaccine is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against 158P3D2-associated disease.

Alternatively, a composition typically comprising transfecting agents is used for the administration of a nucleic acid-based vaccine in accordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native 158P3D2 Sequences

A native 158P3D2 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. The “relatively short” regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, “nested” epitopes can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopes from 158P3D2 antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally, such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup(s) that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native 158P3D2, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid vaccine compositions.

Related to this embodiment, computer programs are available in the art which can be used to identify in a target sequence, the greatest number of epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

The 158P3D2 peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer that expresses 158P3D2 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 158P3D2 as well as tumor-associated antigens that are often expressed with a target cancer associated with 158P3D2 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 158P3D2. Such an analysis can be performed in a manner described by Ogg et al., Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, 158P3D2 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 158P3D2 peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′ triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive non-diseased donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the 158P3D2 epitope, and thus the status of exposure to 158P3D2, or exposure to a vaccine that elicits a protective or therapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 158P3D2-associated disease or who have been vaccinated with a 158P3D2 vaccine.

For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 158P3D2 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific ⁵¹Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of ⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100×[(experimental release−spontaneous release)/maximum release−spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is <25% of maximum release for all experiments.

The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 158P3D2 or a 158P3D2 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptide of the invention, whole 158P3D2 antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for ³H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of ³H-thymidine incorporation in the presence of antigen divided by the ³H-thymidine incorporation in the absence of antigen.

Example 29 Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.

The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.

Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials in Patients Expressing 158P3D2

Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 158P3D2. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 158P3D2, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, e.g., by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows:

The studies are performed in multiple centers. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects (severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and represent diverse ethnic backgrounds. All of them have a tumor that expresses 158P3D2.

Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of 158P3D2-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that used to confirm the efficacy of a DNA vaccine in transgenic mice, such as described above in the Example entitled “The Plasmid Construct and the Degree to Which It Induces Immunogenicity,” can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled “Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 158P3D2 is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells (DC)

Vaccines comprising peptide epitopes of the invention can be administered using APCs, or “professional” APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 158P3D2 protein from which the epitopes in the vaccine are derived.

For example, a cocktail of epitope-comprising peptides is administered ex vivo to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides, and prior to reinfusion into patients, the DC are washed to remove unbound peptides.

As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2-50×10⁶ DC per patient are typically administered, larger number of DC, such as 10⁷ or 10⁸ can also be provided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after treatment with an agent such as Progenipoietin™ are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 10⁸ to 10¹⁰. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin™ mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5×10⁶ DC, then the patient will be injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DC mobilized by an agent such as Progenipoietin™ is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 158P3D2 antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.

Example 33 An Alternative Method of Identifying and Confirming Motif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 158P3D2. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al., J. Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can then be transfected with nucleic acids that encode 158P3D2 to isolate peptides corresponding to 158P3D2 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.

As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.

Example 34 Complementary Polynucleotides

Sequences complementary to the 158P3D2-encoding sequences or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 158P3D2. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of 158P3D2. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 158P3D2-encoding transcript.

Example 35 Purification of Naturally-Occurring or Recombinant 158P3D2 Using 158P3D2-Specific Antibodies

Naturally occurring or recombinant 158P3D2 is substantially purified by immunoaffinity chromatography using antibodies specific for 158P3D2. An immunoaffinity column is constructed by covalently coupling anti-158P3D2 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing 158P3D2 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 158P3D2 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/158P3D2 binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.

Example 36 Identification of Molecules which Interact with 158P3D2

158P3D2, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 158P3D2, washed, and any wells with labeled 158P3D2 complex are assayed. Data obtained using different concentrations of 158P3D2 are used to calculate values for the number, affinity, and association of 158P3D2 with the candidate molecules.

Example 37 In Vivo Assay for 158P3D2 Tumor Growth Promotion

In Vivo Assay of 3T3 Cell Growth by Recombinant Expression of 158P3D2.

To address the determination of 158P3D2 to accelerate the growth of non-tumorigenic cells in an in vivo mouse model, non-transformed 3T3 cells are prepared by infection with either a virus containing an empty vector control (Neo gene alone) or with a vector containing the 158P3D2 full-length gene. 3T3 cells are selected for survival in G-418, and expression of 158P3D2 confirmed by Northern blot analysis. To assess the growth of these cells, 1×10⁶ 158P3D2 expressing 3T3 cells or 1×10⁶ Neo control are mixed with Matrigel®, then injected intratibially or subcutaneously in SCID mice and allowed to grow for 30 days. The growth of these cells is assessed on day 30 by visual inspection and by necropsy. The 158P3D2 expressing 3T3 cells show a potent effect in comparison to the 3T3-Neo cells, indicating that the 158P3D2 protein enhanced the growth of the cells in Matrigel®. 158P3D2 promotes the growth of non-tumorigenic cells and provides a growth advantage in vivo that mimics the role of this protein in human malignancies.

Example 38 158P3D2 Monoclonal Antibody-Mediated Inhibition of Bladder, Lung Colon and Breast and Other Tumors In Vivo

The significant expression of 158P3D2 in cancer tissues, together with its restrictive expression in normal tissues makes 158P3D2 a good target for antibody therapy. Similarly, 158P3D2 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-158P3D2 MAbs in human bladder cancer xenograft mouse models is evaluated by using recombinant cell lines such as J82-158P3D2 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1): p. 16-23), as well as human bladder xenograft models (SCaBER).

Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic bladder cancer xenograft model. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality (see below), as appreciated in the art. Anti-158P3D2 MAbs inhibit formation of bladder xenografts. Anti-158P3D2 MAbs retard the growth of established orthotopic tumors and prolong survival of tumor-bearing mice. MAb effects on tumor growth in mouse models support the utility of anti-158P3D2 MAbs in the treatment of local and advanced stages of bladder cancer (see, e.g., Saffran, D., 2001, et al., PNAS 10:1073-1078).

Administration of the anti-158P3D2 MAbs leads to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. Therefore, 158P3D2 is an attractive target for immunotherapy, and anti-158P3D2 MAbs have therapeutic potential for the treatment of local and metastatic cancer. This example demonstrates that unconjugated 158P3D2 monoclonal antibodies are effective to inhibit the growth of human bladder tumor xenografts grown in SCID mice; accordingly, a combination of such efficacious MAbs is also effective.

MAb-Toxin Conjugates:

Another embodiment of MAb therapy is through the use of toxin conjugation of MAbs for targeted delivery of cytotoxic agents to cells expressing the protein target. Major advances have been made in the clinical application of MAb toxin conjugates with the development of Mylotarg for acute myeloid leukemia (Bross, P. F., et al., 2001, Clin. Cancer Res. 7:1490-1496). Mylotarg is a humanized MAb directed to CD33 which is conjugated to a highly potent DNA-alkylating agent (calichemicin) via an acid labile hydrazone bond (Hamann, P. R., et al., 2002, Bioconjug. Chem. 13:40-46; ibid., 13:47-58). Additional toxins for MAb conjugation in development include maytansinoid, doxorubicin, taxoids and the potent synthetic dolastatin 10 analogs auristatin E and monomethylauristatin E (Doronina, S. O., et al., 2003, Nature Biotech. 21:778-784; Ross, S., et al., 2002, Cancer Res. 62:2546-2553; Francisco, J. A., et el., 2003, Blood 102: 1458-1465; Mao, W., et al., 2004, Cancer Res. 64:781-788). Such applications have potential to deliver a cytotoxic agent to cells expressing the protein target of the MAb. Internalization of the target protein upon MAb binding is important for toxin delivery, and the mechanism spares the non-targeted tissues from the potentially harmful effects of the cytotoxic agent.

158P3D2 MAbs conjugated to toxins are used to induce cell killing in vitro using established protocols for cytotoxicity assays and clonogenic assays (Doronina, S. O., et al., 2003, Nature Biotech. 21:778-784; Mao, W., et al., 2004, Cancer Res. 64:781-788). Toxin conjugated anti-158P3D2 MAbs induce cytotoxicity of cells expressing endogenous 158P3D2 (SCaBER cells) and recombinant 158P3D2 (PC3-158P3D2, 3T3-158P3D2, Rat-1-158P3D2 and B300.19-158P3D2). This methodology allows confirmation that the toxin conjugated MAb is functional against cells expressing the 158P3D2 protein on their surface versus those that do not express the target.

The MAb toxin conjugates are tested for their ability to inhibit tumor growth in vivo. Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic bladder cancer xenograft model, a mouse lung cancer xenograft model, or mouse colon or breast cancer xenograft model. Administration of the anti-158P3D2 MAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 158P3D2 is an attractive target for immunotherapy and demonstrate the therapeutic potential of toxin-conjugated anti-158P3D2 MAbs for the treatment of local and metastatic cancer. This example demonstrates that toxin-conjugated 158P3D2 monoclonal antibodies are effective to inhibit the growth of human bladder, lung, breast and colon tumor xenografts grown in SCID mice; accordingly, a combination of such efficacious MAbs is also effective. The methodology allows the targeted delivery of a cytotoxin using a plasma stable linker in a MAb-toxin conjugate. Such a mechanism of action reduces the potential harmful effects of the toxin on non-targeted tissues.

Tumor Inhibition Using Multiple Unconjugated or Toxin-Conjugated 158P3D2 MAbs

Materials and Methods

158P3D2 Monoclonal Antibodies:

Monoclonal antibodies were raised against 158P3D2 as described in the Example entitled “Generation of 158P3D2 Monoclonal Antibodies (MAbs).” The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 158P3D2. Epitope mapping data for the anti-158P3D2 MAbs, as determined by ELISA and Western analysis, recognize epitopes on the 158P3D2 protein. Immunohistochemical analysis of bladder cancer tissues and cells with these antibodies is performed.

The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at −20° C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, Calif.). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of SCaBER or J82-158P3D2 tumor xenografts.

The MAbs to 158P3D2 are conjugated to various different toxins (listed above) using any of a variety of methods described elsewhere in the art (Hamann, P. R., et al., 2002, Bioconjug. Chem. 13:40-46; ibid., 13:47-58; Doronina, S. O., et al., 2003, Nature Biotech. 21:778-784; Ojima, I., et al. 2002, J. Med. Chem. 45:5620-5623; Dubowchik, G. M., et al., 2002, Bioconjug. Chem. 13:855-869; King, H. D., 2002, J. Med. Chem 45:4336-4343; Ross, S., et al., 2002, Cancer Res. 62:2546-2553; Francisco, J. A., et el., 2003, Blood 102:1458-1465 Mao, W., et al., 2004, Cancer Res. 64:781-788).

Cell Lines

The bladder carcinoma cell lines, J82 and SCaBER, as well as the fibroblast line NIH 3T3 (American Type Culture Collection) are maintained in media supplemented with L-glutamine and 10% FBS. J82-158P3D2 and 3T3-158P3D2 cell populations are generated by retroviral gene transfer as described in Hubert, R. S., et al., Proc. Natl. Acad. Sci. USA, 1999, 96(25): 14523.

Xenograft Mouse Models

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ cancer cells mixed at a 1:1 dilution with Matrigel® (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant protein not expressed in human cells. Tumor sizes are determined by caliper measurements, and the tumor volume is calculated as: Length×Width×Height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed.

Orthotopic injections are performed under anesthesia by using ketamine/xylazine. For bladder orthotopic studies, an incision is made through the abdomen to expose the bladder, and tumor cells (5×10⁵) mixed with Matrigel® are injected into the bladder wall in a 10-μl volume. To monitor tumor growth, mice are palpated and blood is collected on a weekly basis to measure BTA levels. For prostate orthopotic models, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. Tumor cells, e.g. SCaBER cells (5×10⁵) mixed with Matrigel® are injected into the bladder in a 10-μl volume (Yoshida Y et al, Anticancer Res. 1998, 18:327; Ahn et al, Tumor Biol. 2001, 22:146). The mice are segregated into groups for the appropriate treatments, with anti-158P3D2 or control MAbs being injected i.p.

Anti-158P3D2 MAbs Inhibit Growth of 158P3D2-Expressing Xenograft-Cancer Tumors

The effect of anti-158P3D2 MAbs on tumor formation is tested on the growth and progression of bladder cancer xenografts using SCaBER and J82-158P3D2 orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse bladder, and prostate, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem., 1994. 56(1): p. 4-8). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of MAbs on clinically relevant end points.

Accordingly, tumor cells are injected into the mouse bladder, or lung, and 2 days later, the mice are segregated into two groups and treated with either: a) 200-500 μg of anti-158P3D2 MAb, or b) PBS three times per week for two to five weeks.

A major advantage of the orthotopic cancer models is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an antibody against a tumor-specific cell-surface protein such as anti-cytokeratin 20 for bladder cancer models (Lin S et al, Cancer Detect Prev. 2001; 25:202).

Mice bearing established orthotopic tumors are administered 1000 μg injections of either anti-158P3D2 MAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their bladders, livers, bone and lungs are analyzed for the presence of tumor cells by IHC analysis.

Anti-158P3D2 antibodies inhibit the formation of tumors, retard the growth of already established tumors, and prolong the survival of treated mice. Moreover, anti-158P3D2 MAbs demonstrate a dramatic inhibitory effect on the spread of local bladder tumors to distal sites, even in the presence of a large tumor burden. Thus, anti-158P3D2 MAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.

Example 39 Therapeutic and Diagnostic Use of Anti-158P3D2 Antibodies in Humans

Anti-158P3D2 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-158P3D2 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 158P3D2 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-158P3D2 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-158P3D2 mAb specifically binds to carcinoma cells. Thus, anti-158P3D2 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 158P3D2. Shedding or release of an extracellular domain of 158P3D2 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 158P3D2 by anti-158P3D2 antibodies in serum and/or urine samples from suspect patients.

Anti-158P3D2 antibodies that specifically bind 158P3D2 are used in therapeutic applications for the treatment of cancers that express 158P3D2. Anti-158P3D2 antibodies are used as an unconjugated modality and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-158P3D2 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled “158P3D2 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo”). Either conjugated and unconjugated anti-158P3D2 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas Through Use of Human Anti-158P3D2 Antibodies In Vivo

Antibodies are used in accordance with the present invention which recognize an epitope on 158P3D2, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 158P3D2 expression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these indications, three clinical approaches are successfully pursued.

Adjunctive therapy: In adjunctive therapy, patients are treated with anti-158P3D2 antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-158P3D2 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-158P3D2 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).

Monotherapy: In connection with the use of the anti-158P3D2 antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.

Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I¹³¹, Y⁹⁰) to anti-158P3D2 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing 158P3D2. In connection with the use of the anti-158P3D2 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a (¹¹¹In)-158P3D2 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 158P3D2 (by analogy see, e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified.

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-158P3D2 antibodies can be administered with doses in the range of 5 to 400 mg/m², with the lower doses used, e.g., in connection with safety studies. The affinity of anti-158P3D2 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti-158P3D2 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-158P3D2 antibodies can be lower, perhaps in the range of 50 to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery of anti-158P3D2 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-158P3D2 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anti-158P3D2 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 158P3D2 expression levels in their tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 158P3D2. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-158P3D2 antibodies are found to be safe upon human administration.

Example 41 Human Clinical Trial Adjunctive Therapy with Human Anti-158P3D2 Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-158P3D2 antibody in connection with the treatment of a solid tumor, e.g., a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-158P3D2 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-158P3D2 antibody with dosage of antibody escalating from approximately about 25 mg/m² to about 275 mg/m² over the course of the treatment in accordance with the following schedule:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 mg/m² 75 mg/m² 125 mg/m² 175 mg/m² 225 mg/m² 275 mg/m² Chemotherapy + + + + + + (standard dose)

Patients are closely followed for one-week following each administration of antibody and chemotherapy. In particular, patients are assessed for the safety concerns mentioned above: (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 158P3D2. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.

The anti-158P3D2 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.

Example 42 Human Clinical Trial Monotherapy with Human Anti-158P3D2 Antibody

Anti-158P3D2 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-158P3D2 antibodies.

Example 43 Human Clinical Trial Diagnostic Imaging with Anti-158P3D2 Antibody

Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-158P3D2 antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.

Example 44 158P3D2 Functional Assays

158P3D2 protein, and variants thereof, is a member of a family of related proteins, the ferlins. This family of membrane proteins is characterized by the presence of intracellular C2 domains, so named by their homology to a conserved protein kinase C (PKC) motif. The canonical C2 domain is a 130 amino acid long Ca²⁺ dependent membrane targeting module that is found in proteins involved in signal transduction or membrane trafficking (Rizo, J. and Sudhof, T. C., J. Biol. Chem. 273, 15879-82 (1998)). The function of the C2 domain amongst the >100 proteins identified to date varies between these proteins, however a common feature is that the C2 domain has been shown to bind to phospholipids, particularly phosphatidylserine and phosphatidylcholine. In some cases, the C2 domain may not bind to Ca²⁺ or to phospholipids but rather to other proteins (Rizo, J. and Sudhof, T. C., J. Biol. Chem. 273, 15879-82 (1998)). 158P3D2, and variants thereof, are Ca²⁺ binding proteins with the capacity to bind to both phospholipids and to proteins. The different variants of 158P3D2, which express different numbers of C2 domains, have different functions with respect to the unique combinations of expressed C2 regions.

Dysferlin is a member of this family of C2 containing proteins that has a function in muscle membrane repair. Human mutation of dysferlin leads to specific autosomal recessive muscular dystrophies (limb-girdle MD type 2B and Miyoshi myopathy) (reviewed in Bansal, D. and Campbell, K. P., Trends in Cell Biol. 14, 206-213). Dysferlin is localized in the plasma membrane of cells where it interacts with annexin A1 and A2, and is also found in vesicles. Membrane disruption (for example in muscle) causes an increase of localized Ca²⁺ at the wound site and an accumulation of vesicles containing dysferlin. The dysferlin protein facilitates both docking and fusion of the vesicles with the plasma membrane through interaction with the annexins and/or other membrane-associated proteins. Fusion between the repair vesicles and the plasma membrane seals the wound (Bansal, D. and Campbell, K. P., Trends in Cell Biol. 14, 206-213).

158P3D2 protein, and variants thereof, functions in a similar fashion as dysferlin by inducing repair of cellular plasma membranes following their disruption. Given the high rate of cell division and stress conditions such as hypoxia and reduced nutrient supply during tumor formation, membrane repair becomes a critical component of tumor survival. Expression of 158P3D2, and variants thereof, on tumors provides an advantage for such cells to grow under stressful conditions such as hypoxia or nutrient deprivation.

The C2 domain of the lipid phosphatase/tumor suppressor PTEN is regulated by threonine phosphorylation (Raftopolou, M., et al., 2004, Science, 303, 1179-81). This phosphorylation event inhibits cell migration independent of the lipid phosphatase activity, which may relate to the tumor suppressive activity of PTEN. However, given the regulation of C2-induced function by phosphorylation, the status of that phosphorylation event alters the migratory capacity of the cell. 158P3D2 protein, and variants thereof, reside in the plasma membrane of tumor cells as C2-containing regulators of cell migration due to alterations in the phosphorylation status of 158P3D2. Upon phosphorylation of 158P3D2, the C2 domains influence the migratory capacity of 158P3D2-positive tumor cells, conferring an advantage for them to migrate to distal sites to seek secondary growth (metastasis). 158P3D2, and variants thereof, also bind to signal transduction proteins, providing important signaling cascades for tumor cells that confer a growth advantage and increased capacity for cell migration and adhesion. Such advantages are key elements for increased survival and metastasis for bladder, lung, colon and breast cancer cells.

Enhanced proliferation and entry into S-phase of tumor cells relative to normal cells is a hallmark of the cancer cell phenotype. To address the effect of expression of 158P3D2 on the proliferation rate of normal cells, two rodent cell lines (3T3 and Rat-1) are infected with virus containing the 158P3D2 gene and stable cells expressing 158P3D2 antigen are derived, as well as empty vector control cells expressing the selection marker neomycin (Neo). The cells are grown overnight in 0.5% FBS and then compared to cells treated with 10% FBS. The cells are evaluated for proliferation at 18-96 hr post-treatment by a ³H-thymidine incorporation assay and for cell cycle analysis by a BrdU incorporation/propidium iodide staining assay. Rat-1 cells expressing the 158P3D2 antigen grow effectively in low serum concentrations (0.1%) compared to the Rat-1-Neo cells. Similar data are obtained for the 3T3 cells expressing 158P3D2 versus Neo only. To assess cell proliferation by another methodology, the cells are stained with BrdU and propidium iodide. Briefly, cells are labeled with 10 μM BrdU, washed, trypsinized and fixed in 0.4% paraformaldehyde and 70% ethanol. Anti-BrdU-FITC (Pharmigen) is added to the cells, the cells are washed and then incubated with 10 μg/ml propidium iodide for 20 min prior to washing and analysis for fluorescence at 488 nm. An increase in labeling of cells in S-phase (DNA synthesis phase of the cell cycle) in 3T3 cells that express the 158P3D2 protein is observed relative to control cells. This confirms the results of those measured by ³H-thymidine incorporation. Accordingly, 158P3D2 expressing cells have increased potential for growth as tumor cells in vivo during stress, including nutrient deprivation, hypoxia or reduced osmolarity.

Example 45 158P3D2 RNA Interference (RNAi)

RNA interference (RNAi) technology is implemented to a variety of cell assays relevant to oncology. RNAi is a post-transcriptional gene silencing mechanism activated by double-stranded RNA (dsRNA). RNAi induces specific mRNA degradation leading to changes in protein expression and subsequently in gene function. In mammalian cells, these dsRNAs called short interfering RNA (siRNA) have the correct composition to activate the RNAi pathway targeting for degradation, specifically some mRNAs. See, Elbashir S. M., et al., Duplexes of 21-nucleotide RNAs Mediate RNA interference in Cultured Mammalian Cells, Nature 411(6836):494-8 (2001). Thus, RNAi technology is used successfully in mammalian cells to silence targeted genes.

Loss of cell proliferation control is a hallmark of cancerous cells; thus, assessing the role of 158P3D2 in cell survival/proliferation assays is relevant. Accordingly, RNAi was used to investigate the function of the 158P3D2 antigen. To generate siRNA for 158P3D2, algorithms were used that predict oligonucleotides that exhibit the critical molecular parameters (G:C content, melting temperature, etc.) and have the ability to significantly reduce the expression levels of the 158P3D2 protein when introduced into cells. Accordingly, one targeted sequence for the 158P3D2 siRNA is: 5′CCTCGGCAGCCAATCAGCTAT 3′ (SEQ ID NO: 104) (oligo 158P3D2.b). In accordance with this Example, 158P3D2 siRNA compositions are used that comprise siRNA (double stranded, short interfering RNA) that correspond to the nucleic acid ORF sequence of the 158P3D2 protein or subsequences thereof. Thus, siRNA subsequences are used in this manner are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35 contiguous RNA nucleotides in length. These siRNA sequences are complementary and non-complementary to at least a portion of the mRNA coding sequence. In a preferred embodiment, the subsequences are 19-25 nucleotides in length, most preferably 21-23 nucleotides in length. In preferred embodiments, these siRNA achieve knockdown of 158P3D2 antigen in cells expressing the protein and have functional effects as described below.

The selected siRNA (158P3D2.b oligo) was tested in numerous cell lines in the thymidine incorporation/proliferation assay (measures ³H-Thy uptake and incorporation into DNA). Moreover, this 158P3D2.b oligo achieved knockdown of 158P3D2 antigen in cells expressing the protein and had functional effects as described below using the following protocols.

Mammalian siRNA transfections: The day before siRNA transfection, the different cell lines were plated in media (RPMI 1640 with 10% FBS w/o antibiotics) at 2×10³ cells/well in 80 μl (96 well plate format) for the survival/MTS assay. In parallel with the 158P3D2 specific siRNA oligo, the following sequences were included in every experiment as controls: a) Mock transfected cells with Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) and annealing buffer (no siRNA); b) Luciferase-4 specific siRNA (targeted sequence: 5′-AAGGGACGAAGACGAACACUUCTT-3′) (SEQ ID NO: 105); and, c) Eg5 specific siRNA (targeted sequence: 5′-AACTGAAGACCTGAAGACAATAA-3′) (SEQ ID NO: 106). SiRNAs were used at 10 nM and 1 μg/ml Lipofectamine 2000 final concentration.

The procedure was as follows: The siRNAs were first diluted in OPTIMEM (serum-free transfection media, Invitrogen) at 0.1 uM μM (10-fold concentrated) and incubated 5-10 min RT. Lipofectamine 2000 was diluted at 10 μg/ml (10-fold concentrated) for the total number transfections and incubated 5-10 minutes at room temperature (RT). Appropriate amounts of diluted 10-fold concentrated Lipofectamine 2000 were mixed 1:1 with diluted 10-fold concentrated siRNA and incubated at RT for 20-30″ (5-fold concentrated transfection solution). 20 μls of the 5-fold concentrated transfection solutions were added to the respective samples and incubated at 37° C. for 96 hours before analysis.

³H-Thymidine incorporation assay: The proliferation assay is a ³H-thymidine incorporation method for determining the proliferation of viable cells by uptake and incorporation of label into DNA.

The procedure was as follows: Cells growing in log phase are trypsinized, washed, counted and plated in 96-well plates at 1000-4000 cells/well in 10% FBS. After 4-8 hrs, the media is replaced. The cells are incubated for 24-72 hrs, pulsed with ³H-Thy at 1.5 μCi/ml for 14 hrs, harvested onto a filtermat and counted in scintillation cocktail on a Microbeta trilux or other counter.

To address the validation of the 158P3D2 siRNA in reducing the expression of 158P3D2 protein in cells, Cos-1 cells were transfected with pcDNA.3 vector expressing a Myc/His-tagged version of 158P3D2 alone (LF2k) or together with siRNA for either CT1 (bacterial negative control) or 158P3D2 oligo (158P3D2.b). For additional control, a mock transfection was also included (No DNA). Western blot analysis using an antibody to the Myc tag on 158P3D2 protein showed that the 158P3D2 siRNA significantly reduced the expression level of 158P3D2 protein (FIG. 30). These data show that the specific 158P3D2.b siRNA will have utility to probe the function of 158P3D2 protein in cells.

In order to address the function of 158P3D2 in cells, 158P3D2 was silenced by transfecting the endogenously expressing 158P3D2 cell lines (SCaBER, a bladder cancer cell line) with the 158P3D2 specific siRNA (158P3D2.b) along with negative siRNA controls (Luc4, targeted sequence not represented in the human genome) and a positive siRNA control (targeting Eg5) (See FIG. 4). SCaBER cells were shown to express 158P3D2 by Northern blot of total cellular RNA. The results indicated that when these cells were treated with siRNA specifically targeting the 158P3D2 mRNA, the resulting “158P3D2 deficient cells” showed diminished cell proliferation as measured by this assay (see oligo 158P3D2.b treated cells). This effect is likely caused by an active induction of apoptosis. The reduced viability is measured by the decreased uptake of labeled thymidine.

As control, Cos-1 cells, a cell line with no detectable expression of 158P3D2 mRNA or protein (by Western blot), was also treated with the panel of siRNAs (including oligo 158P3D2.b) and no phenotype was observed (FIG. 4). This result reflects the fact that the specific protein knockdown in the SCaBER cells is not a function of general toxicity, since the Cos-1 cells did not respond to the 158P3D2.b oligo. The differential response of the two cell lines to the Eg5 control is a reflection of differences in levels of cell transfection and responsiveness of the cell lines to oligo treatment (FIG. 4).

Together, these data indicate that 158P3D2, and variants thereof, play important roles in the proliferation of cancer cells and that the lack of 158P3D2 clearly decreases the survival potential of these cells. It is to be noted that 158P3D2 is constitutively expressed in many tumor cell lines. 158P3D2 serves a role in malignancy; it expression is a primary indicator of disease, where such disease is often characterized by high rates of uncontrolled cell proliferation and diminished apoptosis. Correlating cellular phenotype with gene knockdown following RNAi treatments is important, and allows one to draw valid conclusions and rule out toxicity or other non-specific effects of these reagents. To this end, assays to measure the levels of expression of both protein and mRNA for the target after RNAi treatments are important, including Western blotting, FACS staining with antibody, immunoprecipitation, Northern blotting or RT-PCR (Taqman or standard methods). Any phenotypic effect of the siRNAs in these assays should be correlated with the protein and/or mRNA knockdown levels in the same cell lines. Knockdown of 158P3D2 is achieved using the 158P3D2.b oligo as measured by Western blotting and RT-PCR analysis.

Another method to analyze 158P3D2 related cell proliferation is performing clonogenic assays. In these assays, a defined number of cells are plated onto the appropriate matrix and the number of colonies formed after a period of growth following siRNA treatment is counted.

In 158P3D2 cancer target validation, complementing the cell survival/proliferation analysis with apoptosis and cell cycle profiling studies are considered. The biochemical hallmark of the apoptotic process is genomic DNA fragmentation, an irreversible event that commits the cell to die. A method to observe fragmented DNA in cells is the immunological detection of histone-complexed DNA fragments by an immunoassay (i.e. cell death detection ELISA) which measures the enrichment of histone-complexed DNA fragments (mono- and oligo-nucleosomes) in the cytoplasm of apoptotic cells. This assay does not require pre-labeling of the cells and can detect DNA degradation in cells that do not proliferate in vitro (i.e. freshly isolated tumor cells).

The most important effector molecules for triggering apoptotic cell death are caspases. Caspases are proteases that when activated cleave numerous substrates at the carboxy-terminal site of an aspartate residue mediating very early stages of apoptosis upon activation. All caspases are synthesized as pro-enzymes and activation involves cleavage at aspartate residues. In particular, caspase 3 seems to play a central role in the initiation of cellular events of apoptosis. Assays for determination of caspase 3 activation detect early events of apoptosis. Following RNAi treatments, Western blot detection of active caspase 3 presence or proteolytic cleavage of products (i.e. PARP) found in apoptotic cells further support an active induction of apoptosis. Because the cellular mechanisms that result in apoptosis are complex, each has its advantages and limitations. Consideration of other criteria/endpoints such as cellular morphology, chromatin condensation, membrane blebbing, apoptotic bodies help to further support cell death as apoptotic. Since not all the gene targets that regulate cell growth are anti-apoptotic, the DNA content of permeabilized cells is measured to obtain the profile of DNA content or cell cycle profile. Nuclei of apoptotic cells contain less DNA due to the leaking out to the cytoplasm (sub-G1 population). In addition, the use of DNA stains (i.e., propidium iodide) also differentiates between the different phases of the cell cycle in the cell population due to the presence of different quantities of DNA in G0/G1, S and G2/M. In these studies the subpopulations can be quantified.

For the 158P3D2 gene, RNAi studies facilitate the understanding of the contribution of the gene product in cancer pathways. Such active RNAi molecules have use in identifying assays to screen for mAbs that are active anti-tumor therapeutics. Further, siRNA are administered as therapeutics to cancer patients for reducing the malignant growth of several cancer types, including those listed in Table 1. When 158P3D2 (and variants) plays a role in cell survival, cell proliferation, tumorigenesis, or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 46 Homology Comparison of 158P3D2 to Known Sequences

The 158P3D2 v.17 protein has 2036 amino acids with a calculated molecular weight of 227.6 kDa and a pI of 5.64. 158P3D2 is predicted to be a predominantly cytoplasmic protein with plasma membrane association (PSORT-II). 158P3D2 contains a single transmembrane region from amino acids 2000-2022 with high probability that the amino-terminus resides outside, consistent with the topology of a type I transmembrane protein. Based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel, TMBASE-A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993), 158P3D2 contains a primary transmembrane region from amino acids 2003-2020 (contiguous amino acids with values greater than 0 on the plot have high probability of being transmembrane regions) with an orientation in which the amino terminus resides inside and the carboxyl terminus outside (type II). Another transmembrane algorithm indicated that 158P3D2 contains a transmembrane domain from amino acids 2003-2022, with the N-terminus oriented intracellularly consistent with a type II topology. The transmembrane prediction algorithms are accessed through the ExPasy molecular biology server.

By use of the PubMed website of the N.C.B.I., it was found at the protein level that 158P3D2 v.17 shows 60% homology and 40% identity with human otoferlin, a member of the ferlin family of plasma membrane proteins. Further, 158P3D2 v.17 shows 50% homology and 30% identity with dysferlin, another member of the ferlin family, and 80% homology and 75% identity with the murine gene Fer-1-like 4.

The ferlins are a family of transmembrane proteins that have function in membrane trafficking, including the repair of cell membranes. Mutation of human otoferlin leads to a specific form of nonsyndromic autosomal recessive deafness (DFNB9) and mutations in dysferlin lead to two subtypes of muscular dystrophies (reviewed in Bansal, D. and Campbell, K. P., Trends in Cell Biol. 14, 206-213). The major feature of the ferlin family includes multiple C2 domains (conserved PKC homologous region) that function in both Ca²⁺ dependent and Ca²⁺ independent phospholipid binding, as well as protein binding. The mechanism of action for dysferlin includes the repair of muscle cell membrane disruptions through dysferlin-containing cell vesicles. Such vesicles are tethered to the site of membrane tears (where Ca²⁺ concentrations are increased) via dysferlin molecules that interact with plasma membrane associated annexin A1 and annexin A2 molecules. The vesicles provide the lipid bilayer material to seal the wound.

158P3D2 associates with cell vesicles and the plasma membrane thereby providing a means for tumor cells to repair membranes during tumor growth and metastasis. Such a functional advantage can be exemplified by the increased stress that tumors experience, including increased hypoxia, decreased nutrition and increases in free radical formation. These stresses can alter membrane integrity, thereby increasing the need for robust plasma membrane repair mechanisms. In addition, the C2 domains of 158P3D2, and variants thereof, regulate migration of tumor cells expressing this protein. The regulation of the C2 domains occurs through the phosphorylation of threonine residues (Raftopolou, M., et al., 2004, Science, 303, 1179-81) and modulates the ability of the expressing cells to migrate. This signal transduction property of the 158P3D2 protein expressed in tumor cells enhances their ability to migrate during metastases, facilitates their homing to distal sites (lymph nodes), and promotes interactions with other cells during the formation of tumor masses. Further, the C2 domains of 158P3D2 play a role in membrane trafficking though interaction with Ca²⁺, phosphatidylserine and phosphatidylcholine (Rizo, J. and Sudhof, T. C., J. Biol. Chem. 273, 15879-82 (1998). These interactions are crucial for subsequent membrane metabolism and interaction. Taken together, the 158P3D2 protein significantly promotes unregulated growth of cancer cells, contributing to their viability and metastatic advantage in vivo.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

Tables

XV.)

TABLE I TISSUES THAT EXPRESS 158P3D2 WHEN MALIGNANT. Prostate Bladder Kidney Colon Ovary Lung Breast Pancreas Stomach Cervix Lymph node Uterus

XVI.)

TABLE II AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cys cysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamine R Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asn asparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid E Glu glutamic acid G Gly glycine

TABLE III Amino Acid Substitution Matrix Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. (See world wide web URL ikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L M N P Q R S T V W Y . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

TABLE IV HLA Class I/II Motifs/Supermotifs

TABLE IV (A) HLA Class I Supermotifs/Motifs POSITION POSITION POSITION C Terminus 2 (Primary 3 (Primary (Primary  Anchor) Anchor) Anchor) SUPERMOTIF A1 TI LVMS FWY A2 LIVM ATQ IV MATL A3 VSMA TLI RK A24 YF WIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYL WMIVA B44 E D FWYLIMVA B58 ATS FWY LIVMA B62 QL IVMP FWY MIVLA MOTIFS A1 TSM Y A1 DE AS Y A2.1 LM VQIAT V LIMAT A3 LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YFWM FLIW A*3101 MVT ALIS R K A*3301 MVALF IST RK A*6801 AVT MSLI RK B*0702 P LMF WYAIV B*3501 P LMFWY IVA B51 P LIVF WYAM B*5301 P IMFWY ALV B*5401 P ATIV LMFWY

Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.

TABLE IV (B) HLA Class II Supermotif 1 6 9 W, F, Y, V, I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C) HLA Class II Motifs 1° MOTIFS anchor 1 2 3 4 5 1° anchor 6 7 8 9 DR4 preferred FMYLI M T I VSTCPA MH MH VW LIM deleterious W R W DE DR1 preferred MFLIV PAMQ VMATS M AVM WY PLIC deleterious C CH FD CWD GDE D DR7 preferred MFLIV M W A IVMSAC M IV WY TPL deleterious C G GRD N G DR3 MOTIFS 1° 2 3 1° 5 1° anchor 6 anchor 1 anchor 4 Motif a LIVMFY D preferred Motif b LIVMF DNQEST KRH preferred AY DR MFLIV VMSTA Supermotif WY CPLI Italicized residues indicate less preferred or “tolerated” residues

TABLE IV (D) HLA Class I Supermotifs SUPER- POSITION: MOTIFS 1 2 3 4 5 6 7 8 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY A2 1° Anchor 1° Anchor LIVMATQ LIVMAT A3 Preferred 1° Anchor YFW YFW YFW P 1° Anchor VSMATLI (4/5) (3/5) (4/5) (4/5) RK deleterious DE (3/5); DE P (5/5) (4/5) A24 1° Anchor 1° Anchor YFWIVLMT FIYWLM B7 Preferred FWY (5/5) 1° Anchor FWY FWY 1°Anchor LIVM P (4/5) (3/5) VILFMWYA (3/5) deleterious DE (3/5); DE G QN DE P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5); A(3/5); QN(3/5) B27 1° Anchor 1°Anchor RHK FYLWMIVA B44 1° Anchor 1° Anchor ED FWYLIMVA B58 1° Anchor 1° Anchor ATS FWYLIVMA B62 1° Anchor 1° Anchor QLIVMP FWYMIVLA Italicized residues indicate less preferred or “tolerated” residues

TABLE IV (E) HLA Class I Motifs POSITION 9 or C- C- 1 2 3 4 5 6 7 8 terminus terminus A1 preferred GFYW 1°Anchor DEA YFW P DEQN YFW 1°Anchor 9-mer STM Y deleterious DE RHKLIV A G A MP A1 preferred GRHK ASTCLIVM 1°Anchor GSTC ASTC LIVM DE 1°Anchor 9-mer DEAS Y deleterious A RHKDEP DE PQN RHK PG GP YFW A1 preferred YFW 1°Anchor DEAQN A YFWQN PASTC GDE P 1°Anchor 10-mer STM Y deleterious GP RHKGLI DE RHK QNA RHKYFW RHK A VM A1 preferred YFW STCLIVM 1°Anchor A YFW PG G YFW 1°Anchor 10-mer DEAS Y deleterious RHK RHKDEP P G PRHK QN YFW A2.1 preferred YFW 1°Anchor YFW STC YFW A P 1°Anchor 9-mer LMIVQAT VLIMAT deleterious DEP DERKH RKH DERKH A2.1 preferred AYFW 1°Anchor LVIM G G FYWL 1°Anchor 10-mer LMIVQAT VIM VLIMAT deleterious DEP DE RKHA P RKH DER RKH KH A3 preferred RHK 1°Anchor YFW PRHKY A YFW P 1°Anchor LMVISA FW KYRHFA TFCGD deleterious DEP DE A11 preferred A 1°Anchor YFW YFW A YFW YFW P 1°Anchor VTLMIS KRYH AGNCDF deleterious DEP A G A24 preferred YFWRHK 1°Anchor STC YFW YFW 1°Anchor 9-mer YFWM FLIW deleterious DEG DE G QNP DER G AQN HK A24 preferred 1°Anchor P YFWP P 1°Anchor 10-mer YFWM FLIW deleterious GDE QN RHK DE A QN DEA A3101 preferred RHK 1°Anchor YFW P YFW YFW AP 1°Anchor MVTALIS RK deleterious DEP DE ADE DE DE DE A3301 preferred 1°Anchor YFW AYFW 1°Anchor MVALFI RK ST deleterious GP DE A1 preferred GFYW 1°Anchor DEA YFW P DEQN YFW 1°Anchor 9-mer STM Y deleterious DE RHKLIV A G A MP A1 preferred GRHK ASTCLIVM 1°Anchor GSTC ASTC LIVM DE 1°Anchor 9-mer DEAS Y deleterious A RHKDEP DE PQN RHK PG GP YFW A6801 preferred YFWSTC 1°Anchor YFWLI YFW P 1°Anchor AVTMSLI VM RK deleterious GP DEG RHK A B0702 preferred RHKFWY 1°Anchor RHK RHK RHK RHK PA 1°Anchor P LMFWY AIV deleterious DEQNP DEP DE DE GDE QN DE B3501 preferred FWYLI 1°Anchor FWY FWY 1°Anchor VM P LMFWY IVA deleterious AGP G G B51 preferred LIVMF 1°Anchor FWY STC FWY G FWY 1°Anchor WY P LIVFWY AM deleterious AGPDE DE G DEQN GDE RHKSTC B5301 preferred LIVMF 1°Anchor FWY STC FWY LIVMF FWY 1°Anchor WY P WY IMFWY ALV deleterious AGPQN G RHKQN DE B5401 preferred FWY 1°Anchor FWYLIVM LIVM ALIVM FWY 1°Anchor P AP ATIVLM FWY deleterious GPQNDE GDESTC RHKDE DE QNDGE DE

TABLE IV (F) Summary of HLA-supertypes Overall phenotypic frequencies of HLA-supertypes in different ethnic populations Specificity Phenotypic frequency Supertype Position 2 C-Terminus Caucasian N.A. Black Japanese Chinese Hispanic Average B7 P AILMVFWY 43.2 55.1 57.1 43.0 49.3 49.5 A3 AILMVST RK 37.5 42.1 45.8 52.7 43.1 44.2 A2 AILMVT AILMVT 45.8 39.0 42.4 45.9 43.0 42.2 A24 YF (WIVLMT) FI (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0 B44 E (D) FWYLIMVA 43.0 21.2 42.9 39.1 39.0 37.0 A1 TI (LVMS) FWY 47.1 16.1 21.8 14.7 26.3 25.2 B27 RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4 B62 QL (IVMP) FWY (MIV) 12.6 4.8 36.5 25.4 11.1 18.1 B58 ATS FWY (LIV) 10.0 25.1 1.6 9.0 5.9 10.3

TABLE IV (G) Calculated population coverage afforded by different HLA-supertype combinations Phenotypic frequency HLA-supertypes Caucasian N.A Blacks Japanese Chinese Hispanic Average A2, A3 and B7 83.0 86.1 87.5 88.4 86.3 86.2 A2, A3, B7, 99.5 98.1 100.0 99.5 99.4 99.3 A24, B44 and A1 A2, A3, B7, 99.9 99.6 100.0 99.8 99.9 99.8 A24, B44, A1, B27, B62, and B 58 Motifs indicate the residues defining supertype specificites. The motifs incorporate residues determined on the basis of published data to be recognized by multiple alleles within the supertype. Residues within brackets are additional residues also predicted to be tolerated by multiple alleles within the supertype.

TABLE V Frequently Occurring Motifs avrg. % Name identity Description Potential Function zf-C2H2 34% Zinc finger, C2H2 Nucleic acid-binding protein functions as type transcription factor, nuclear location probable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase, generate superoxide terminal)/b6/petB Ig 19% Immunoglobulin domains are one hundred amino acids long and domain include a conserved intradomain disulfide bond. WD40 18% WD domain, G- tandem repeats of about 40 residues, each beta repeat containing a Trp-Asp motif. Function in signal transduction and protein interaction PDZ 23% PDZ domain may function in targeting signaling molecules to sub-membranous sites LRR 28% Leucine Rich short sequence motifs involved in protein- Repeat protein interactions Pkinase 23% Protein kinase conserved catalytic core common to both domain serine/threonine and tyrosine protein kinases containing an ATP binding site and a catalytic site PH 16% PH domain pleckstrin homology involved in intracellular signaling or as constituents of the cytoskeleton EGF 34% EGF-like domain 30-40 amino-acid long found in the extracellular domain of membrane-bound proteins or in secreted proteins Rvt 49% Reverse transcriptase (RNA-dependent DNA polymerase) Ank 25% Ank repeat Cytoplasmic protein, associates integral membrane proteins to the cytoskeleton Oxidored_q1 32% NADH- membrane associated. Involved in proton Ubiquinone/plastoquinone translocation across the membrane (complex I), various chains Efhand 24% EF hand calcium-binding domain, consists of a12 residue loop flanked on both sides by a 12 residue alpha-helical domain Rvp 79% Retroviral aspartyl Aspartyl or acid proteases, centered on a protease catalytic aspartyl residue Collagen 42% Collagen triple extracellular structural proteins involved in helix repeat (20 formation of connective tissue. The sequence copies) consists of the G-X-Y and the polypeptide chains forms a triple helix. Fn3 20% Fibronectin type III Located in the extracellular ligand-binding domain region of receptors and is about 200 amino acid residues long with two pairs of cysteines involved in disulfide bonds 7tm_1 19% 7 transmembrane seven hydrophobic transmembrane regions, receptor (rhodopsin with the N-terminus located extracellularly family) while the C-terminus is cytoplasmic. Signal through G proteins

TABLE VI Motif-bearing subsequences and Post- Translational Modifications of 158P3D2 Phosphorylation sites predicted: Pos.  Context Phosphoserine predictions 73 SRVFSLRPL (SEQ ID NO: 107) 107 NLQVSPIQV (SEQ ID NO: 108) 128 TGAWSEEDF (SEQ ID NO: 109) 173 RLARSLGQQ (SEQ ID NO: 110) 206 GVMFSPLKS (SEQ ID NO: 111) 210 SPLKSRARA (SEQ ID NO: 112) 268 QRGTSCPFY (SEQ ID NO: 113) 320 LTPPSPKAF (SEQ ID NO: 114) 380 KVTLSVRAR (SEQ ID NO: 115) 460 YVRVSFLGQ (SEQ ID NO: 116) 469 EGETSVSAE (SEQ ID NO: 117) 471 ETSVSAEAA (SEQ ID NO: 118) 484 NEQLSFVEL (SEQ ID NO: 119) 522 LRRISHPGR (SEQ ID NO: 120) 624 HLDASPGAE (SEQ ID NO: 121) 679 SQPISFEIS (SEQ ID NO: 122) 683 SFEISIGRA (SEQ ID NO: 123) 916 DVLFSVVEE (SEQ ID NO: 124) 982 GLPSSLHRD (SEQ ID NO: 125) 1012 DSGLSDPFA (SEQ ID NO: 126) 1127 ELDYSGRLE (SEQ ID NO: 127) 1136 PSVPSEVEP (SEQ ID NO: 128) 1151 VEPHSGRLS (SEQ ID NO: 129) 1155 SGRLSLPPN (SEQ ID NO: 130) 1208 EVLASYRES (SEQ ID NO: 131) 1212 SYRESPNFT (SEQ ID NO: 132) 1380 DPGDSDGVN (SEQ ID NO: 133) 1405 KGTVSPKKA (SEQ ID NO: 134) 1419 IYNRSLKEE (SEQ ID NO: 135) 1519 YVVVSAGRE (SEQ ID NO: 136) 1585 NRFYSHHRA (SEQ ID NO: 137) 1595 CGLASQYEV (SEQ ID NO: 138) 1652 LPPGSSSPT (SEQ ID NO: 139) 1654 PGSSSPTVA (SEQ ID NO: 140) 1699 YHPHSPGLL (SEQ ID NO: 141) 1733 RQPISYELR (SEQ ID NO: 142) 1809 EREVSVWRR (SEQ ID NO: 143) 1814 VWRRSGPFA (SEQ ID NO: 144) 1841 YDRISANDF (SEQ ID NO: 145) 1975 LEKPSRPKT (SEQ ID NO: 146) Phosphothreonine predictions 29 FRGFTQKTR (SEQ ID NO: 147) 261 QRRVTATQR (SEQ ID NO: 148) 263 RVTATQRGT (SEQ ID NO: 149) 267 TQRGTSCPF (SEQ ID NO: 150) 338 TRIGTFRMD (SEQ ID NO: 151) 367 DPRDTRAGT (SEQ ID NO: 152) 601 LSRLTRKKK (SEQ ID NO: 153) 613 RRDQTPKAV (SEQ ID NO: 154) 704 AGEGTEGAA (SEQ ID NO: 155) 828 RRTMTRPNA (SEQ ID NO: 156) 1022 VLISTQCQT (SEQ ID NO: 157) 1026 TQCQTTRVL (SEQ ID NO: 158) 1033 VLEQTLSPL (SEQ ID NO: 159) 1223 VRHLTVVFK (SEQ ID NO: 160) 1412 KAVATLKIY (SEQ ID NO: 161) 1527 ERQDTKERY (SEQ ID NO: 162) 1557 ETELTVAVF (SEQ ID NO: 163) 1643 KVFLTPPET (SEQ ID NO: 164) 1743 VIWNTEDVV (SEQ ID NO: 165) 1804 DYLPTEREV (SEQ ID NO: 166) 1941 VYILTGKVE (SEQ ID NO: 167) 1952 FELLTVEEA (SEQ ID NO: 168) 1979 SRPKTSFNW (SEQ ID NO: 169) Phosphotyrosine predictions 52 RWPHYGAPL (SEQ ID NO: 170) 456 LVEPYVRVS (SEQ ID NO: 171) 542 WVPLYGSPP (SEQ ID NO: 172) 990 DDFSYFQLR (SEQ ID NO: 173) 1095 MEDPYQRPE (SEQ ID NO: 174) 1243 PEQPYLQPP (SEQ ID NO: 175) 1515 KADPYVVVS (SEQ ID NO: 176) 1531 TKERYIPKQ (SEQ ID NO: 177) 1602 EVDGYNAWR (SEQ ID NO: 178) 1629 PAPEYRAGA (SEQ ID NO: 179) 1763 SSDIYVKSW (SEQ ID NO: 180) N-glycosylation sites 67 NCSR (SEQ ID NO: 181) 1214 NFTE (SEQ ID NO: 182) 1417 NRSL (SEQ ID NO: 183) O-glycosylation sites 1403 T 1647 T 1656 T Dileucine motifs LL at 290 LL at 291 LL at 406 LL at 407 LL at 438 LL at 571 LL at 572 LL at 643 LL at 658 LL at 714 LL at 840 LL at 847 LL at 855 LL at 874 LL at 971 LL at 1041 LL at 1184 LL at 1320 LL at 1333 LL at 1491 C2 domains aa2-96 aa230-355 aa420-530 aa992-1100 aa1492-1591 aa1735-1865 Coiled-coil regions aa168-186 aa1895-1922 Interaction motifs and specific kinase phosphorylation sites proteins interaction motif 2 RRTMTRP (SEQ ID NO: 184) FHA domain interaction motif 1, threonine phosphorylation is required TPKA (SEQ ID NO: 185) TEGA (SEQ ID NO: 186) TWRL (SEQ ID NO: 187) TSEL (SEQ ID NO: 188) TAPL (SEQ ID NO: 189) TLKI (SEQ ID NO: 190) TRPL (SEQ ID NO: 191) Motif found in p53 family members which confers binding to the N-terminal domain of MDM2 FGPAWVPL (SEQ ID NO: 192) Class III PDZ domains binding motif DENL (SEQ ID NO: 193) FELI (SEQ ID NO: 194) RDSL (SEQ ID NO: 195) NEGV (SEQ ID NO: 196) VEEL (SEQ ID NO: 197) EELL (SEQ ID NO: 198) PENV (SEQ ID NO: 199) EDFL (SEQ ID NO: 200) EEQL (SEQ ID NO: 201) EEEL (SEQ ID NO: 202) AERL (SEQ ID NO: 203) VERL (SEQ ID NO: 204) LEVL (SEQ ID NO: 205) PDVL (SEQ ID NO: 206) QDVL (SEQ ID NO: 207) PDLL (SEQ ID NO: 208) GELI (SEQ ID NO: 209) VEVL (SEQ ID NO: 210) SEVL (SEQ ID NO: 211) TELV (SEQ ID NO: 212) EEDI (SEQ ID NO: 213) PEEL (SEQ ID NO: 214) SDGV (SEQ ID NO: 215) EDWL (SEQ ID NO: 216) SEAV (SEQ ID NO: 217) PETL (SEQ ID NO: 218) PEHV (SEQ ID NO: 219) PEDL (SEQ ID NO: 220) FELL (SEQ ID NO: 221) PEPL (SEQ ID NO: 222) Src-family Src Homology 2 (SH2) domains binding motif. YQPP (SEQ ID NO: 223) This is the motif recognized by class I SH3 domains RAEPEPP (SEQ ID NO: 224) RLEPSVP (SEQ ID NO: 225) This is the motif recognized by those SH3 domains with a non-canonical class I recognition specificity LPPPMLP (SEQ ID NO: 226) PMLPPAP (SEQ ID NO: 227) RAEPEPP (SEQ ID NO: 228) AEGPEIP (SEQ ID NO: 229) PENVLAP (SEQ ID NO: 230) AEEPQPP (SEQ ID NO: 231) EPQPPLP (SEQ ID NO: 232) RLEPSVP (SEQ ID NO: 233) LLEVEQP (SEQ ID NO: 234) QDLPEQP (SEQ ID NO: 235) DMMPKGP (SEQ ID NO: 236) LYHPHSP (SEQ ID NO: 237) PQDVPAP (SEQ ID NO: 238) QDVPAPP (SEQ ID NO: 239) DIKPRQP (SEQ ID NO: 240) LEKPSRP (SEQ ID NO: 241) This is the motif recognized by those SH3 domains with a non-canonical class II  recognition specificity KPPLKKLP (SEQ ID NO: 242) Major TRAF2-binding consensus motif. Members of the tumor necrosis factor receptor (TNFR) superfamily initiate intracellular signaling by recruiting the C-domain of the TNFR-associated factors (TRAFs) through their cytoplasmic tails. PEEE (SEQ ID NO: 243) PPEE (SEQ ID NO: 244) PEEE (SEQ ID NO: 245) SLQE (SEQ ID NO: 246) TVEE (SEQ ID NO: 247) TRAF6 binding site. Members of the tumor  necrosis factor receptor (TNFR) superfamily initiate intracellular signaling by recruiting the C-domain of the TNFR-associated factors (TRAFs) through their cytoplasmatic tails. GVPAERPWA (SEQ ID NO: 248) ARPEEEKEE (SEQ ID NO: 249) DPPEEEGEM (SEQ ID NO: 250) PDPEELDWG (SEQ ID NO: 251) PPLP is the motif recognized by WW domains of    Group II PPLP (SEQ ID NO: 252) GSK3 phosphorylation recognition site GATGAWS (SEQ ID NO: 253) MFSPLKS (SEQ ID NO: 254) RDTRAGT (SEQ ID NO: 255) VASQPIS (SEQ ID NO: 256) PISFEIS (SEQ ID NO: 257) IQSLMLT (SEQ ID NO: 258) PHSGRLS (SEQ ID NO: 259) LASYRES (SEQ ID NO: 260) RESPNFT (SEQ ID NO: 261) FLTPPET (SEQ ID NO: 262) PGSSSPT (SEQ ID NO: 263) KPSRPKT (SEQ ID NO: 264) PKA phosphorylation site RDT RDS RLT RGS RRT RLS RES RFT Site phosphorylated by the Polo-like-kinase EATM (SEQ ID NO: 265) DPTV (SEQ ID NO: 266) EISI (SEQ ID NO: 267) DHTW (SEQ ID NO: 268) EPSA (SEQ ID NO: 269) EPSV (SEQ ID NO: 270) Proline-Directed Kinase (e.g. MAPK) phosphoryla- tion site in higher eukaryotes. LQVSPIQ (SEQ ID NO: 271) VMFSPLK (SEQ ID NO: 272) LYGSPPG (SEQ ID NO: 273) RDQTPKA (SEQ ID NO: 274) LDASPGA (SEQ ID NO: 275) YRESPNF (SEQ ID NO: 276) GTVSPKK (SEQ ID NO: 277) VFLTPPE (SEQ ID NO: 278) GSSSPTV (SEQ ID NO: 279) HPHSPGL (SEQ ID NO: 280) SH-PTP2 and phospholipase C-gamma Src Homology 2 (SH2) domains binding motif. aa249-252 aa456-459 aa1515-1518 CK1 phosphorylation site aa1009-1012 aa234-237 aa484-487 aa760-763 CK2 phosphorylation site aa1804-1807 PKB Phosphorylation site aa1522-1528

TABLE VII Search Peptides 158P3D2 v.1, 9-mers, 10-mers and 15-mers (SEQ ID NO: 281) MWIDIFPQDV PAPPPVDIKP RQPISYELRV VIWNTEDVVL DDENPLTGEM SSDIYVKSWV 60 KGLEHDKQET DVHFNSLTGE GNFNWRFVFR FDYLPTEREV SVWRRSGPFA LEEAEFRQPA 120 VLVLQVWDYD RISANDFLGS LELQLPDMVR GARGPELCSV QLARNGAGPR CNLFRCRRLR 180 GWWPVVKLKE AEDVEREAQE AQAGKKKRKQ RRRKGRPEDL EFTDMGGNVY ILTGKVEAEF 240 ELLTVEEAEK RPVGKGRKQP EPLEKPSRPK TSFNWFVNPL KTFVFFIWRR YWRTLVLLLL 300 VLLTVFLLLV FYTIPGQISQ VIFRPLHK 328 158P3D2 v.2A, 9-mers, 10-mers and 15-mers (SEQ ID NO: 282) MDDPGDSDGV NLISMVGEIQ DQGEAEVKGT VSPKKAVATL KIYNRSLEEE FNHFEDWLNV 60 FPLYRGQGGQ DGGGEEEGSG HLVGKFKGSF LIYPESEAVL FSEPQISRGI PQNRPIKLLV 120 RVYVVKATNL APADPNGKAD PYVVVSAGRE RQDTKERYIP KQLNPIFGEI LELSISLPAE 180 TELTVAVFEH DLVGSDDLIG ETHIDLENRF YSHHRANCGL ASQYEVWVQQ GPQEPF 236 158P3D2 v.3 9-mers aa 95-111 PTEREVSVRRRSGPFAL (SEQ ID NO: 283) 10-mers aa 94-112 LPTEREVSVRRRSGPFALE (SEQ ID NO: 284) 15-mers aa 89-117 FRFDYLPTEREVSVRRRSGPFALEEAEFR (SEQ ID NO: 285) 158P3D2 v.4 9-mers aa 94-110 LPTEREVSIWRRSGPFA (SEQ ID NO: 286) 10-mers aa 93-111 YLPTEREVSIWRRSGPFAL (SEQ ID NO: 287) 15-mers aa 88-116 VFRFDYLPTEREVSIWRRSGPFALEEAEF (SEQ ID NO: 288) 158P3D2 v.5A (BCP2A) ORF: 849-1385 9-mers LVLQVWDYTASLPMTSLDPWSCSYQTWCVGP (SEQ ID NO: 289) GAPSSALCSWPAMGPGRGAICFAAAA 10-mers VLVLQVWDYTASLPMTSLDPWSCSYQTWCVGP (SEQ ID NO: 290) GAPSSALCSWPAMGPGRGAICFAAAA 15-mers FRQPAVLVLQVWDYTASLPMTSLDPWSCSYQT (SEQ ID NO: 291) WCVGPGAPSSALCSWPAMGPGRGAICFAAAA 158P3D2 v.10 9-mers aa 50-66 MSSDIYVKRWVKGLEHD (SEQ ID NO: 292) 10-mers aa 49-67 EMSSDIYVKRWVKGLEHDK (SEQ ID NO: 293) 15-mers aa 44-72 NPLTGEMSSDIYVKRWVKGLEHDKQETDV (SEQ ID NO: 294) 158P3D2 v.12 9-mers aa 273-287 FNWFVNPLNTFVFFIWR (SEQ ID NO: 295) 10-mers aa 272-288 SFNWFVNPLNTFVFFIWRR (SEQ ID NO: 296) 15-mers aa 267-293 SRPKTSFNWFVNPLNTFVFFIWRRYWRTL (SEQ ID NO: 297) 158P3D2 v.13 9-mers aa 274-288 NWFVNPLKAFVFFIWRR (SEQ ID NO: 298) 10-mers aa 273-287 FNWFVNPLKAFVFFIWRRY (SEQ ID NO: 299) 15-mers aa 268-294 RPKTSFNWFVNPLKAFVFFIWRRYWRTLV (SEQ ID NO: 300) Combination of v12 and v13 9-mers aa 273-287 NWFVNPLNAFVFFIWR (SEQ ID NO: 301) 10-mers aa 272-288 FNWFVNPLNAFVFFIWRR (SEQ ID NO: 302) 15-mers aa 267-293 RPKTSFNWFVNPLNAFVFFIWRRYWRTL (SEQ ID NO: 303) v.14 ORF: 65-4246 Frame +2 Part A 9-mers ELVRHLTVDLPEQPYL (SEQ ID NO: 304) 10-mers ELVRHLTVDLPEQPYLQ (SEQ ID NO: 305) 15-mers ESPNFTELVRHLTVDLPEQPYLQPPLSI (SEQ ID NO: 306) Part B 9-mers MVGEIQDQDLQQVPEGRI (SEQ ID NO: 307) 10-mers SMVGEIQDQDLQQVPEGRI (SEQ ID NO: 308) 15-mers GVNLISMVGEIQDQDLQQVPEGRI (SEQ ID NO: 309) v.15 ORF: 65-3502 Frame +2 Part A 9-mers KLRFLAEEHNFDEDEM (SEQ ID NO: 310) 10-mers AKLRFLAEEHNFDEDEM (SEQ ID NO: 311) 15-mers AKKLLAKLRFLAEEHNFDEDEMDDPGDS (SEQ ID NO: 312) Part B 9-mers LVRVYVVKLRNLCKIQGHEDFCLFSAATNLAPAD (SEQ ID NO: 313) 10-mers LLVRVYVVKLRNLCKIQGHEDFCLFSAATNLAPADP (SEQ ID NO: 314) 15-mers NRPIKLLVRVYVVKLRNLCKIQGHEDFCLFSAATNL (SEQ ID NO: 315) APADPNGKAD Part C 9-mers GLASQYEVWVQQGPQEPF (SEQ ID NO: 316) 10-mers CGLASQYEVWVQQGPQEPF (SEQ ID NO: 317) 15-mers HHRANCGLASQYEVWVQQGPQEPF (SEQ ID NO: 318) v.16 ORF: 65-6037 Frame +2 1990 AA Part A 9-mers GSKVFLTPPETLPPVASGDPEEAQALLV (SEQ ID NO: 319) 10-mers GSKVFLTPPETLPPVASGDPEEAQALLV (SEQ ID NO: 320) 15-mers GSKVFLTPPETLPPVASGDPEEAQALLV (SEQ ID NO: 321) Part B 9-mers VKLKEAEDGKVEAEFE (SEQ ID NO: 322) 10mers VVKLKEAEDGKVEAEFEL (SEQ ID NO: 323) 15-mers RGWWPVVKLKEAEDGKVEAEFELLTVEE (SEQ ID NO: 324) v.17 ORF: 65-6175 Frame +2 9-mers, 10-mers and 15-mers (SEQ ID NO: 325) MALTVSVQRL TGLTGTHDRQ VKLTFRGFTQ KTRKIHCGPE ADIGELFRWP HYGAPLAGEC 60 LSVQVVNCSR VFSLRPLGTL VISLQQLQNA GHLVLREALV DENLQVSPIQ VELDLKYQPP 120 EGATGAWSEE DFGAPIQDSF ELIIPNVGFQ ELEPGEAQLE RRAVALGRRL ARSLGQQDDE 180 ENELELELEQ DLDDEPDVEL SGVMFSPLKS RARALAHGDP FQVSRAQDFQ VGVTVLEAQK 240 LVGVNINPYV AVQVGGQRRV TATQRGTSCP FYNEYFLFEF HDTRLRLQDL LLEITVSGVG 300 VTSVLQRRGD EKAAGLTPPS PKAFHSQTLP FMATRIGTFR MDLGIILDQP DGQFYQRWVP 360 LHDPRDTRAG TKGFIKVTLS VRARGDLPPP MLPPAPGHCS DIEKNLLLPR GVPAERPWAR 420 LRVRLYRAEG LPALRLGLLG SLVRALHDQR VLVEPYVRVS FLGQEGETSV SAEAAAPEWN 480 EQLSFVELFP PLTRSLRLQL RDDAPLVDAA LATHVPDLRR ISHPGRAAGF NPTFGPAWVP 540 LYGSPPGAGL RDSLQGLNEG VGQGIWFRGR LLLAVSMQVL EGRAEPEPPQ AQQGSTLSRL 600 TRKKKKKARR DQTPKAVPQH LDASPGAEGP EIPRAMEVEV EELLPLPENV LAPCEDFLLF 660 GVLFEATMID PTVASQPISF EISIGRAGRL EEQLGRGSRA GEGTEGAAVE AQPLLGARPE 720 EEKEEEELGT HAQRPEPMDG SGPYFCLPLC HCKPCMHVWS CWEDHTWRLQ SSNCVRKVAE 780 RLDQGLQEVE RLQRKPGPGA CAQLKQALEV LVAGSRQFCH GAERRTMTRP NALDRCRGKL 840 LVHSLNLLAK QGLRLLRGLR RRNVQKKVAL AKKLLAKLRF LAEEPQPPLP DVLVWMLSGQ 900 RRVAWARIPA QDVLFSVVEE ERGRDCGKIQ SLMLTAPGAA PGEVCAKLEL FLRLGLGKQA 960 KACTSELPPD LLPEPSAGLP SSLHRDDFSY FQLRAHLYQA RGVLAADDSG LSDPFARVLI 1020 STQCQTTRVL EQTLSPLWDE LLVFEQLIVD GRREHLQEEP PLVIINVFDH NKFGPPVFLG 1080 RALAAPRVKL MEDPYQRPEL QFFPLRKGPW AAGELIAAFQ LIELDYSGRL EPSVPSEVEP 1140 QDLAPLVEPH SGRLSLPPNV CPVLREFRVE VLFWGLRGLG RVHLLEVEQP QVVLEVAGQG 1200 VESEVLASYR ESPNFTELVR HLTVVFKDTA PLFHPQDLPE QPYLQPPLSI LVIERRAFGH 1260 TVLVGSHIVP HMLRFTFRGH EDPPEEEGEM EETGDMMPKG PQGQKSLDPF LAEAGISRQL 1320 LKPPLKKLPL GGLLNQGPGL EEDIPDPEEL DWGSKYYASL QELQGQHNFD EDEMDDPGDS 1380 DGVNLISMVG EIQDQGEAEV KGTVSPKKAV ATLKIYNRSL KEEFNHFEDW LNVFPLYRGQ 1440 GGQDGGGEEE GSGHLVGKFK GSFLIYPESE AVLFSEPQIS RGIPQNRPIK LLVRVYVVKA 1500 TNLAPADPNG KADPYVVVSA GRERQDTKER YIPKQLNPIF GEILELSISL PAETELTVAV 1560 FDHDLVGSDD LIGETHIDLE NRFYSHHRAN CGLASQYEVD GYNAWRDAFW PSQILAGLCQ 1620 RCGLPAPEYR AGAVKVGSKV FLTPPETLPP GSSSPTVASG DPEEAQALLV LRRWQEMPGF 1680 GIQLVPEHVE TRPLYHPHSP GLLQGSLHMW IDIFPQDVPA PPPVDIKPRQ PISYELRVVI 1740 WNTEDVVLDD ENPLTGEMSS DIYVKSWVKG LEHDKQETDV HFNSLTGEGN FNWRFVFRFD 1800 YLPTEREVSV WRRSGPFALE EAEFRQPAVL VLQVWDYDRI SANDFLGSLE LQLPDMVRGA 1860 RGPELCSVQL ARNGAGPRCN LFRCRRLRGW WPVVKLKEAE DVEREAQEAQ AGKKKRKQRR 1920 RKGRPEDLEF TDMGGNVYIL TGKVEAEFEL LTVEEAEKRP VGKGRKQPEP LEKPSRPKTS 1980 FNWFVNPLKT FVFFIWRRYW RTLVLLLLVL LTVFLLLVFY TIPGQISQVI FRPLHK 2036 v.18 ORF: 2932-4764 Frame +1 610 aa Part A 9-mers MCKRRWHWPRSSWQNCAFWLRRHPGQPLVRSVPSWS (SEQ ID NO: 326) SSCGWAWASKPRPAPLSCPRICCPSPQPGCPPAYTG TVLEQTLSP 10-mers MCKRRWHWPRSSWQNCAFWLRRHPGQPLVRSVPSWS (SEQ ID NO: 327) SSCGWAWASKPRPAPLSCPRICCPSPQPGCPPAYTG TVLEQTLSPL 15-mers MCKRRWHWPRSSWQNCAFWLRRHPGQPLVRSVPSWS (SEQ ID NO: 328) SSCGWAWASKPRPAPLSCPRICCPSPQPGCPPAYTG TVLEQTLSPLWDELL Part B 9-mers GISRQLLKHNFDEDEM (SEQ ID NO: 329) 10-mers AGISRQLLKHNFDEDEMD (SEQ ID NO: 330) 15-mers PFLAEAGISRQLLKHNFDEDEMDDPGDS (SEQ ID NO: 331) Part C 9-mers GLASQYEVWVQQGPQEPF (SEQ ID NO: 332) 10-mers CGLASQYEVWVQQGPQEPF (SEQ ID NO: 333) 15-mers HHRANCGLASQYEVWVQQGPQEPF (SEQ ID NO: 334)

TABLE VIII 158P3D2v.1-A1-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 222 FTDMGGNVY 62.500 47 TGEMSSDIY 11.250 219 DLEFTDMGG 4.500 110 ALEEAEFRQ 4.500 237 EAEFELLTV 4.500 247 EAEKRPVGK 3.600 198 AQEAQAGKK 2.700 78 TGEGNFNWR 2.250 259 QPEPLEKPS 2.250 113 EAEFRQPAV 1.800 140 SLELQLPDM 1.800 281 KTFVFFIWR 1.250 303 LTVFLLLVF 1.250 145 LPDMVRGAR 1.250 312 YTIPGQISQ 1.250 69 ETDVHFNSL 1.250 34 NTEDVVLDD 1.125 320 QVIFRPLHK 1.000 166 GAGPRCNLF 1.000 304 TVFLLLVFY 1.000 39 VLDDENPLT 1.000 188 LKEAEDVER 0.900 235 KVEAEFELL 0.900 190 EAEDVEREA 0.900 62 GLEHDKQET 0.900 51 SSDIYVKSW 0.750 2 WIDIFPQDV 0.500 257 RKQPEPLEK 0.500 142 ELQLPDMVR 0.500 283 FVFFIWRRY 0.500 121 VLVLQVWDY 0.500 156 ELCSVQLAR 0.500 154 GPELCSVQL 0.450 97 EREVSVWRR 0.450 242 LLTVEEAEK 0.400 197 EAQEAQAGK 0.400 243 LTVEEAEKR 0.250 90 RFDYLPTER 0.250 49 EMSSDIYVK 0.200 4 DIFPQDVPA 0.200 11 PAPPPVDIK 0.200 123 VLQVWDYDR 0.200 53 DIYVKSWVK 0.200 262 PLEKPSRPK 0.180 75 NSLTGEGNF 0.150 67 KQETDVHFN 0.135 126 VWDYDRISA 0.125 293 RTLVLLLLV 0.125 81 GNFNWRFVF 0.125 277 VNPLKTFVF 0.125 77 LTGEGNFNW 0.125 214 KGRPEDLEF 0.125 270 KTSFNWFVN 0.125 85 WRFVFRFDY 0.125 40 LDDENPLTG 0.125 216 RPEDLEFTD 0.113 298 LLLVLLTVF 0.100 200 EAQAGKKKR 0.100 170 RCNLFRCRR 0.100 109 FALEEAEFR 0.100 276 FVNPLKTFV 0.100 244 TVEEAEKRP 0.090 25 SYELRVVIW 0.090 193 DVEREAQEA 0.090 195 EREAQEAQA 0.090 132 ISANDFLGS 0.075 316 GQISQVIFR 0.075 105 RSGPFALEE 0.075 10 VPAPPPVDI 0.050 71 DVHFNSLTG 0.050 300 LVLLTVFLL 0.050 137 FLGSLELQL 0.050 232 LTGKVEAEF 0.050 294 TLVLLLLVL 0.050 301 VLLTVFLLL 0.050 302 LLTVFLLLV 0.050 227 GNVYILTGK 0.050 297 LLLLVLLTV 0.050 296 VLLLLVLLT 0.050 131 RISANDELG 0.050 308 LLVFYTIPG 0.050 245 VEEAEKRPV 0.045 143 LQLPDMVRG 0.030 24 ISYELRVVI 0.030 201 AQAGKKKRK 0.030 50 MSSDIYVKS 0.030 116 FRQPAVLVL 0.025 46 LTGEMSSDI 0.025 191 AEDVEREAQ 0.025 95 PTEREVSVW 0.022 59 WVKGLEHDK 0.020 179 LRGWWPVVK 0.020 306 FLLLVFYTI 0.020 157 LCSVQLARN 0.020 230 YILTGKVEA 0.020 309 LVFYTIPGQ 0.020 299 LLVLLTVFL 0.020 17 DIKPRQPIS 0.020 295 LVLLLLVLL 0.020 158 CSVQLARNG 0.015

TABLE IX 158P3D2v.1-A1-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 259 QPEPlEKPSR 45.000 276 FVNPlKTFVF 5.000 166 GAGPrCNLFR 5.000 235 KVEAeFELLT 4.500 198 AQEAqAGKKK 2.700 39 VLDDeNPLTG 2.500 303 LTVFlLLVFY 2.500 17 DIKPrQPISY 2.500 222 FTDMgGNVYI 2.500 78 TGEGnFNWRF 2.250 113 EAEFrQPAVL 1.800 46 LTGEmSSDIY 1.250 69 ETDVhFNSLT 1.250 47 TGEMsSDIYV 1.125 140 SLELqLPDMV 0.900 219 DLEFtDMGGN 0.900 190 EAEDvEREAQ 0.900 244 TVEEaEKRPV 0.900 51 SSDIyVKSWV 0.750 67 KQETdVHFNS 0.675 134 ANDFlGSLEL 0.625 120 AVLVlQVWDY 0.500 302 LLTVfLLLVF 0.500 10 VPAPpPVDIK 0.500 95 PTEReVSVWR 0.450 241 ELLTvEEAEK 0.400 312 YTIPgQISQV 0.250 281 KTFVfFIWRR 0.250 145 LPDMvRGARG 0.250 77 LTGEgNFNWR 0.250 12 APPPvDIKPR 0.250 154 GPELcSVQLA 0.225 216 RPEDlEFTDM 0.225 34 NTEDvVLDDE 0.225 25 SYELrVVIWN 0.225 122 LVLQvWDYDR 0.200 231 ILTGkVEAEF 0.200 197 EAQEaQAGKK 0.200 200 EAQAgKKKRK 0.200 100 VSVWrRSGPF 0.150 105 RSGPfALEEA 0.150 319 SQVIfRPLHK 0.150 80 EGNFnWRFVF 0.125 293 RTLVlLLLVL 0.125 297 LLLLvLLTVF 0.100 144 QLPDmVRGAR 0.100 242 LLTVeEAEKR 0.100 193 DVEReAQEAQ 0.090 247 EAEKrPVGKG 0.090 62 GLEHdKQETD 0.090 245 VEEAeKRPVG 0.090 110 ALEEaEFRQP 0.090 237 EAEFeLLTVE 0.090 107 GPFAlEEAEF 0.050 15 PVDIkPRQPI 0.050 304 TVFLlLVFYT 0.050 2 WIDIfPQDVP 0.050 76 SLTGeGNFNW 0.050 307 LLLVfYTIPG 0.050 300 LVLLtVFLLL 0.050 295 LVLLlLVLLT 0.050 301 VLLTvFLLLV 0.050 299 LLVLlTVFLL 0.050 261 EPLEkPSRPK 0.050 277 VNPLkTFVFF 0.050 109 FALEeAEFRQ 0.050 81 GNFNwRFVFR 0.050 296 VLLLlVLLTV 0.050 314 IPGQISQVIF 0.050 226 GGNVyILTGK 0.050 131 RISAnDFLGS 0.050 97 EREVsVWRRS 0.045 239 EFELlTVEEA 0.045 111 LEEAeFRQPA 0.045 41 DDENpLTGEM 0.045 195 EREAqEAQAG 0.045 178 RLRGwWPVVK 0.040 24 ISYElRVVIW 0.030 139 GSLElQLPDM 0.030 318 ISQViFRPLH 0.030 224 DMGGnVYILT 0.025 165 NGAGpRCNLF 0.025 282 TFVFfIWRRY 0.025 280 LKTFvFFIWR 0.025 82 NFNWrFVFRF 0.025 171 CNLFrCRRLR 0.025 126 VWDYdRISAN 0.025 128 DYDRiSANDF 0.025 141 LELQlPDMVR 0.025 35 TEDVvLDDEN 0.025 74 FNSLtGEGNF 0.025 221 EFTDmGGNVY 0.025 294 TLVLlLLVLL 0.020 38 VVLDdENPLT 0.020 142 ELQLpDMVRG 0.020 53 DIYVkSWVKG 0.020 246 EEAEkRPVGK 0.020 187 KLKEaEDVER 0.020 272 SFNWfVNPLK 0.020 298 LLLVlLTVFL 0.020

TABLE X 158P3D2v.1-A0201-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 302 LLTVFLLLV 1033.404 297 LLLLVLLTV 1006.209 286 FIWRRYWRT 440.113 306 FLLLVFYTI 337.376 301 VLLTVFLLL 255.302 299 LLVLLTVFL 199.738 300 LVLLTVFLL 156.843 276 FVNPLKTFV 153.971 296 VLLLLVLLT 107.808 137 FLGSLELQL 98.267 2 WIDIFPQDV 66.867 38 VVLDDENPL 48.205 48 GEMSSDIYV 27.521 31 VIWNTEDVV 27.109 295 LVLLLLVLL 27.042 313 TIPGQISQV 21.996 39 VLDDENPLT 20.776 294 TLVLLLLVL 20.145 230 YILTGKVEA 11.626 144 QLPDMVRGA 9.370 293 RTLVLLLLV 8.221 30 VVIWNTEDV 5.069 141 LELQLPDMV 4.168 236 VEAEFELLT 3.838 178 RLRGWWPVV 3.684 94 LPTEREVSV 3.165 180 RGWWPVVKL 2.662 228 NVYILTGKV 2.532 305 VFLLLVFYT 2.388 279 PLKTFVFFT 2.240 121 VLVLQVWDY 2.185 240 FELLTVEEA 1.853 133 SANDFLGSL 1.382 124 LQVWDYDRI 1.322 224 DMGGNVYIL 1.091 118 QPAVLVLQV 1.044 46 LTGEMSSDI 1.010 83 FNWRFVFRF 0.941 27 ELRVVIWNT 0.733 140 SLELQLPDM 0.731 234 GKVEAEFEL 0.706 55 YVKSWVKGL 0.692 114 AEFRQPAVL 0.630 24 ISYELRVVI 0.623 52 SDIYVKSWV 0.531 62 GLEHDKQET 0.477 177 RRLRGWWPV 0.456 22 QPISYELRV 0.454 298 LLLVLLTVF 0.442 159 SVQLARNGA 0.435 76 SLTGEGNFN 0.410 235 KVEAEFELL 0.390 183 WPVVKLKEA 0.343 269 PKTSFNWFV 0.333 26 YELRVVIWN 0.312 304 TVFLLLVFY 0.305 186 VKLKEAEDV 0.298 223 TDMGGNVYI 0.295 307 LLLVFYTIP 0.219 4 DIFPQDVPA 0.190 165 NGAGPRCNL 0.139 272 SFNWFVNPL 0.130 308 LLVFYTIPG 0.127 225 MGGNVYILT 0.124 10 VPAPPPVDI 0.116 112 EEAEFRQPA 0.113 135 NDFLGSLEL 0.110 143 LQLPDMVRG 0.109 281 KTFVFFIWR 0.106 171 CNLFRCRRL 0.103 8 QDVPAPPPV 0.097 318 ISQVIFRPL 0.090 87 FVFRFDYLP 0.084 86 RFVFRFDYL 0.076 93 YLPTEREVS 0.069 80 EGNFNWRFV 0.064 131 RISANDFLG 0.059 290 RYWRTLVLL 0.057 314 IPGQISQVI 0.047 77 LTGEGNFNW 0.042 79 GEGNFNWRF 0.041 23 PISYELRVV 0.040 70 TDVHFNSLT 0.039 109 FALEEAEFR 0.039 283 FVFFIWRRY 0.038 122 LVLQVWDYD 0.038 106 SGPFALEEA 0.037 68 QETDVHFNS 0.034 168 GPRCNLFRC 0.033 292 WRTLVLLLL 0.031 245 VEEAEKRPV 0.029 319 SQVIFRPLH 0.029 231 ILTGKVEAE 0.029 317 QISQVIFRP 0.027 120 AVLVLQVWD 0.027 215 GRPEDLEFT 0.026 242 LLTVEEAEK 0.025 123 VLQVWDYDR 0.025 16 VDIKPRQPI 0.025 258 KQPEPLEKP 0.024

TABLE XI 158P3D2v.1-A0201-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 301 VLLTvFLLLV 3823.593 296 VLLLlVLLTV 1006.209 298 LLLVlLTVFL 739.032 299 LLVLlTVFLL 484.457 93 YLPTeREVSV 319.939 304 TVFLlLVFYT 177.011 278 NPLKtFVFFI 70.254 294 TLVLlLLVLL 49.134 26 YELRvVIWNT 42.542 286 FIWRrYWRTL 38.130 300 LVLLtVFLLL 22.339 236 VEAEfELLTV 21.680 101 SVWRrSGPFA 19.844 31 VIWNtEDVVL 16.993 38 VVLDdENPLT 16.816 87 FVFRfDYLPT 16.647 117 RQPAvLVLQV 16.219 125 QVWDyDRISA 14.793 123 VLQVwDYDRI 13.036 312 YTIPgQISQV 10.220 295 LVLLlLVLLT 9.433 63 LEHDkQETDV 9.426 21 RQPIsYELRV 7.052 114 AEFRqPAVLV 5.004 271 TSFNwFVNPL 4.510 68 QETDvHFNSL 3.236 29 RVVIwNTEDV 2.982 61 KGLEhDKQET 2.583 79 GEGNfNWRFV 2.529 268 RPKTsFNWFV 2.491 140 SLELqLPDMV 2.181 30 VVIWnTEDVV 2.078 273 FNWFvNPLKT 1.857 222 FTDMgGNVYI 1.466 143 LQLPdMVRGA 1.457 275 WFVNpLKTFV 1.222 139 GSLElQLPDM 1.132 317 QISQvIFRPL 1.116 220 LEFTdMGGNV 1.106 293 RTLVlLLLVL 1.035 51 SSDIyVKSWV 0.999 309 LVFYtIPGQI 0.746 224 DMGGnVYILT 0.605 306 FLLLvFYTIP 0.593 313 TIPGqISQVI 0.588 153 RGPElCSVQL 0.572 235 KVEAeFELLT 0.555 307 LLLVfYTIPG 0.469 297 LLLLvLLTVF 0.442 167 AGPRcNLFRC 0.433 76 SLTGeGNFNW 0.432 120 AVLVlQVWDY 0.416 112 EEAEfRQPAV 0.416 244 TVEEaEKRPV 0.319 91 FDYLpTEREV 0.284 189 KEAEdVEREA 0.277 172 NLFRcRRLRG 0.276 132 ISANdFLGSL 0.269 285 FFIWrRYWRT 0.268 85 WRFVfRFDYL 0.259 1 MWIDIFPQDV 0.256 148 MVRGaRGPEL 0.242 45 PLTGeMSSDI 0.230 39 VLDDeNPLTG 0.208 185 VVKLkEAEDV 0.177 281 KTFVfFIWRR 0.176 151 GARGpELCSV 0.169 47 TGEMsSDIYV 0.160 137 FLGSlELQLP 0.158 37 DVVLdDENPL 0.140 164 RNGAgPRCNL 0.139 231 ILTGkVEAEF 0.127 283 FVFFIWRRYW 0.122 302 LLTVfLLLVF 0.119 121 VLVLqVWDYD 0.116 234 GKVEaEFELL 0.113 258 KQPEpLEKPS 0.108 223 TDMGgNVYIL 0.104 292 WRTLvLLLLV 0.102 305 VFLLlVFYTI 0.087 22 QPISyELRVV 0.086 109 FALEeAEFRQ 0.084 214 KGRPeDLEFT 0.080 276 FVNPlKTFVF 0.071 9 DVPApPPVDI 0.068 7 PQDVpAPPPV 0.062 227 GNVYILTGKV 0.059 308 LLVFyTIPGQ 0.058 290 RYWRtLVLLL 0.057 134 ANDFlGSLEL 0.056 194 VEREaQEAQA 0.051 111 LEEAeFRQPA 0.040 230 YILTgKVEAE 0.039 19 KPRQpISYEL 0.037 105 RSGPfALEEA 0.037 158 CSVQlARNGA 0.032 233 TGKVeAEFEL 0.028 129 YDRIsANDFL 0.028 170 RCNLfRCRRL 0.028 177 RRLRgWWPVV 0.025

TABLE XII 158P3D2v.1-A3-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 281 KTFVFFIWR 54.000 121 VLVLQVWDY 54.000 123 VLQVWDYDR 36.000 49 EMSSDIYVK 27.000 242 LLTVEEAEK 20.000 306 FLLLVFYTI 12.150 53 DIYVKSWVK 9.000 301 VLLTVFLLL 8.100 320 QVIFRPLHK 6.000 298 LLLVLLTVF 4.500 142 ELQLPDMVR 3.600 156 ELCSVQLAR 3.600 316 GQISQVIFR 3.240 59 WVKGLEHDK 3.000 304 TVFLLLVFY 3.000 294 TLVLLLLVL 2.700 224 DMGGNVYIL 2.430 172 NLFRCRRLR 2.000 302 LLTVFLLLV 1.800 279 PLKTFVFFI 1.620 297 LLLLVLLTV 1.350 137 FLGSLELQL 1.200 181 GWWPVVKLK 1.013 299 LLVLLTVFL 0.900 296 VLLLLVLLT 0.900 178 RLRGWWPVV 0.900 300 LVLLTVFLL 0.810 81 GNFNWRFVF 0.540 235 KVEAEFELL 0.540 83 FNWRFVFRF 0.540 303 LTVFLLLVF 0.450 243 LTVEEAEKR 0.450 201 AQAGKKKRK 0.450 227 GNVYILTGK 0.405 62 GLEHDKQET 0.300 273 FNWFVNPLK 0.300 262 PLEKPSRPK 0.300 283 FVFFIWRRY 0.300 101 SVWRRSGPF 0.300 140 SLELQLPDM 0.300 55 YVKSWVKGL 0.270 27 ELRVVIWNT 0.203 222 FTDMGGNVY 0.200 85 WRFVFRFDY 0.180 308 LLVFYTIPG 0.180 198 AQEAQAGKK 0.180 79 GEGNFNWRF 0.162 286 FIWRRYWRT 0.150 232 LTGKVEAEF 0.150 295 LVLLLLVLL 0.135 11 PAPPPVDIK 0.135 21 RQPISYELR 0.120 170 RCNLFRCRR 0.120 31 VIWNTEDVV 0.100 39 VLDDENPLT 0.100 278 NPLKTFVFF 0.090 187 KLKEAEDVE 0.090 231 ILTGKVEAE 0.090 265 KPSRPKTSF 0.090 87 FVFRFDYLP 0.090 110 ALEEAEFRQ 0.090 307 LLLVFYTIP 0.090 38 VVLDDENPL 0.090 166 GAGPRCNLF 0.090 109 FALEEAEFR 0.090 197 EAQEAQAGK 0.090 282 TFVFFIWRR 0.081 179 LRGWWPVVK 0.060 257 RKQPEPLEK 0.060 144 QLPDMVRGA 0.060 268 RPKTSFNWF 0.060 247 EAEKRPVGK 0.060 2 WIDIFPQDV 0.060 46 LTGEMSSDI 0.045 293 RTLVLLLLV 0.045 4 DIFPQDVPA 0.045 77 LTGEGNFNW 0.045 313 TIPGQISQV 0.045 93 YLPTEREVS 0.040 230 YILTGKVEA 0.030 76 SLTGEGNFN 0.030 228 NVYILTGKV 0.030 57 KSWVKGLEH 0.030 276 FVNPLKTFV 0.030 30 VVIWNTEDV 0.030 199 QEAQAGKKK 0.030 69 ETDVHFNSL 0.027 319 SQVIFRPLH 0.027 168 GPRCNLFRC 0.027 124 LQVWDYDRI 0.027 96 TEREVSVWR 0.027 24 ISYELRVVI 0.022 159 SVQLARNGA 0.020 161 QLARNGAGP 0.020 285 FFIWRRYWR 0.018 250 KRPVGKGRK 0.018 214 KGRPEDLEF 0.018 78 TGEGNFNWR 0.018 154 GPELCSVQL 0.018 22 QPISYELRV 0.018

TABLE XIII 158P3D2v.1-A3-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 178 RLRGwWPVVK 90.000 281 KTFVfFIWRR 40.500 187 KLKEaEDVER 18.000 241 ELLTvEEAEK 9.000 299 LLVLlTVFLL 8.100 302 LLTVfLLLVF 6.000 122 LVLQvWDYDR 5.400 120 AVLVlQVWDY 5.400 297 LLLLvLLTVF 4.500 231 ILTGkVEAEF 4.500 242 LLTVeEAEKR 4.000 301 VLLTvFLLLV 2.700 144 QLPDmVRGAR 1.800 319 SQVIfRPLHK 1.800 296 VLLLlVLLTV 1.350 294 TLVLlLLVLL 1.350 10 VPAPpPVDIK 1.350 48 GEMSsDIYVK 1.215 161 QLARnGAGPR 1.200 298 LLLVlLTVFL 0.900 77 LTGEgNFNWR 0.900 276 FVNPlKTFVF 0.900 76 SLTGeGNFNW 0.900 300 LVLLtVFLLL 0.810 123 VLQVwDYDRI 0.600 303 LTVFlLLVFY 0.450 304 TVFLlLVFYT 0.450 81 GNFNwRFVFR 0.360 17 DIKPrQPISY 0.360 166 GAGPrCNLFR 0.360 31 VIWNtEDVVL 0.300 107 GPFAlEEAEF 0.300 46 LTGEmSSDIY 0.300 198 AQEAqAGKKK 0.300 279 PLKTfVFFIN 0.270 278 NPLKtFVFFI 0.243 180 RGWWpVVKLK 0.225 93 YLPTeREVSV 0.200 140 SLELqLPDMV 0.200 172 NLFRcRRLRG 0.200 125 QVWDyDRISA 0.200 307 LLLVfYTIPG 0.180 235 KVEAeFELLT 0.180 96 TEREvSVWRR 0.162 226 GGNVyILTGK 0.135 293 RTLVlLLLVL 0.135 309 LVFYtIPGQI 0.135 224 DMGGnVYILT 0.135 271 TSFNwFVNPL 0.135 313 TIPGqISQVI 0.135 256 GRKQpEPLEK 0.120 87 FVFRfDYLPT 0.100 101 SVWRrSGPFA 0.100 52 SDIYvKSWVK 0.090 295 LVLLlLVLLT 0.090 148 MVRGaRGPEL 0.090 306 FLLLvEYTIP 0.090 45 PLTGeMSSDI 0.090 286 FIWRrYWRTL 0.090 19 KPRQpISYEL 0.081 280 LKTFvFFIWR 0.072 62 GLEHdKQETD 0.060 259 QPEPlEKPSR 0.060 284 VFFIwRRYWR 0.060 196 REAQeAQAGK 0.060 82 NFNWrFVFRF 0.054 141 LELQlPDMVR 0.054 121 VLVLqVWDYD 0.045 308 LLVEyTIPGQ 0.045 12 APPPvDIKPR 0.045 39 VLDDeNPLTG 0.040 84 NWRFvFRFDY 0.036 168 GPRCnLERCR 0.036 117 RQPAvLVLQV 0.036 21 RQPIsYELRV 0.036 312 YTIPgQISQV 0.034 272 SFNWfVNPLK 0.030 58 SWVKgLEHDK 0.030 200 EAQAgKKKRK 0.030 30 VVIWnTEDVV 0.030 283 FVFFiWRRYW 0.030 137 FLGSlELQLP 0.030 222 FTDMgGNVYI 0.030 29 RVVIwNTEDV 0.030 95 PTEReVSVWR 0.030 37 DVVLdDENPL 0.027 78 TGEGnFNWRF 0.027 9 DVPApPPVDI 0.027 317 QISQvIFRPL 0.027 270 KTSFnWFVNP 0.027 246 EEAEkRPVGK 0.027 197 EAQEaQAGKK 0.027 131 RISAnDFLGS 0.024 24 ISYElRVVIW 0.022 261 EPLEkPSRPK 0.020 314 IPGQiSQVIF 0.020 202 QAGKkKRKQR 0.020 89 FRFDyLPTER 0.020 185 VVKLkEAEDV 0.020 316 GQISqVIFRP 0.018

TABLE XIV 158P3D2v.1-A1101-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 320 QVIFRPLHK 6.000 281 KTFVFFIWR 2.400 59 WVKGLEHDK 2.000 316 GQISQVIFR 1.080 198 AQEAQAGKK 0.600 53 DIYVKSWVK 0.480 242 LLTVEEAEK 0.400 21 RQPISYELR 0.360 243 LTVEEAEKR 0.300 201 AQAGKKKRK 0.300 49 EMSSDIYVK 0.240 227 GNVYILTGK 0.180 123 VLQVWDYDR 0.160 257 RKQPEPLEK 0.120 90 RFDYLPTER 0.120 282 TFVFFIWRR 0.120 170 RCNLFRCRR 0.120 285 FFIWRRYWR 0.120 293 RTLVLLLLV 0.090 300 LVLLTVFLL 0.090 273 FNWFVNPLK 0.080 181 GWWPVVKLK 0.060 250 KRPVGKGRK 0.060 109 FALEEAEFR 0.060 247 EAEKRPVGK 0.060 197 EAQEAQAGK 0.060 235 KVEAEFELL 0.060 142 ELQLPDMVR 0.048 156 ELCSVQLAR 0.048 145 LPDMVRGAR 0.040 304 TVFLLLVFY 0.040 82 NFNWRFVFR 0.040 101 SVWRRSGPF 0.040 228 NVYILTGKV 0.040 162 LARNGAGPR 0.040 95 LVLLLLVLL 0.030 77 LTGEGNFNW 0.030 199 QEAQAGKKK 0.030 303 LTVFLLLVF 0.030 38 VVLDDENPL 0.030 30 VVIWNTEDV 0.030 290 RYWRTLVLL 0.024 276 FVNPLKTFV 0.020 179 LRGWWPVVK 0.020 11 PAPPPVDIK 0.020 159 SVQLARNGA 0.020 172 NLFRCRRLR 0.016 204 GKKKRKQRR 0.012 306 FLLLVFYTI 0.012 301 VLLTVFLLL 0.012 121 VLVLQVWDY 0.012 96 TEREVSVWR 0.012 178 RLRGWWPVV 0.012 297 LLLLVLLTV 0.012 294 TLVLLLLVL 0.012 232 LTGKVEAEF 0.010 222 FTDMGGNVY 0.010 55 YVKSWVKGL 0.010 46 LTGEMSSDI 0.010 29 RVVIWNTED 0.009 124 LQVWDYDRI 0.009 270 KTSFNWFVN 0.009 86 RFVFRFDYL 0.009 319 SQVIFRPLH 0.009 302 LLTVFLLLV 0.008 87 FVFRFDYLP 0.008 137 FLGSLELQL 0.008 167 AGPRCNLFR 0.008 31 VIWNTEDVV 0.008 81 GNFNWRFVF 0.007 48 GEMSSDIYV 0.007 208 RKQRRRKGR 0.006 206 KKRKQRRRK 0.006 154 GPELCSVQL 0.006 230 YILTGKVEA 0.006 22 QPISYELRV 0.006 299 LLVLLTVFL 0.006 193 DVEREAQEA 0.006 298 LLLVLLTVF 0.006 265 KPSRPKTSF 0.006 166 GAGPRCNLF 0.006 200 EAQAGKKKR 0.006 175 RCRRLRGWW 0.006 268 RPKTSENWF 0.006 262 PLEKPSRPK 0.004 25 SYELRVVIW 0.004 2 WIDIFPQDV 0.004 78 TGEGNFNWR 0.004 188 LKEAEDVER 0.004 309 LVFYTIPGQ 0.004 118 QPAVLVLQV 0.004 313 TIPGQISQV 0.004 283 FVFFIWRRY 0.004 310 VFYTIPGQI 0.004 140 SLELQLPDM 0.004 131 RISANDELG 0.004 79 GEGNENWRF 0.004 312 YTIPGQISQ 0.003 278 NPLKTFVFF 0.003 120 AVLVLQVWD 0.003

TABLE XV 158P3D2v.1-A1101-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 281 KTFVfFIWRR 2.400 319 SQVIfRPLHK 1.800 122 LVLQvWDYDR 1.200 178 RLRGwWPVVK 1.200 48 GEMSsDIYVK 0.720 198 AQEAqAGKKK 0.300 166 GAGPrCNLFR 0.240 187 KLKEaEDVER 0.240 272 SFNWfVNPLK 0.200 10 VPAPpPVDIK 0.200 77 LTGEgNFNWR 0.200 241 ELLTvEEAEK 0.180 196 REAQeAQAGK 0.180 284 VFFIwRRYWR 0.160 256 GRKQpEPLEK 0.120 29 RVVIwNTEDV 0.090 293 RTLVlLLLVL 0.090 125 QVWDyDRISA 0.080 144 QLPDmVRGAR 0.080 161 QLARnGAGPR 0.080 242 LLTVeEAEKR 0.080 226 GGNVyILTGK 0.060 180 RGWWpVVKLK 0.060 52 SDIYvKSWVK 0.060 120 AVLVlQVWDY 0.060 300 LVLLtVFLLL 0.060 197 EAQEaQAGKK 0.060 276 FVNPlKTFVF 0.060 81 GNFNwRFVFR 0.048 290 RYWRtLVLLL 0.048 101 SVWRrSGPFA 0.040 259 QPEPlEKPSR 0.040 309 LVEYtIPGQI 0.040 21 RQPIsYELRV 0.036 141 LELQlPDMVR 0.036 117 RQPAvLVLQV 0.036 58 SWVKgLEHDK 0.030 200 EAQAgKKKRK 0.030 30 VVIWnTEDVV 0.030 96 TEREvSVWRR 0.024 12 APPPvDIKPR 0.020 148 MVRGaRGPEL  0.020 202 QAGKkKRKQR 0.020 185 VVKLkEAEDV 0.020 95 PTEReVSVWR 0.020 299 LLVLlTVELL 0.018 246 EEAEkRPVGK 0.018 303 LTVFlLLVEY 0.015 312 YTIPgQISQV 0.015 168 GPRCnLFRCR 0.012 235 KVEAeFELLT 0.012 19 KPRQpISYEL 0.012 304 TVFLlLVFYT 0.012 296 VLLLlVLLTV 0.012 107 GPFAlEEAEF 0.012 76 SLTGeGNFNW 0.012 301 VLLTvFLLLV 0.012 268 RPKTsFNWFV 0.012 222 FTDMgGNVYI 0.010 46 LTGEmSSDIY 0.010 37 DVVLdDENPL 0.009 261 EPLEkPSRPK 0.009 278 NPLKtFVFFI 0.009 316 GQISqVIFRP 0.008 280 LKTFvFFIWR 0.008 87 FVFRfDYLPT 0.008 302 LLTVfLLLVF 0.008 89 FRFDyLPTER 0.008 31 VINNtEDVVL 0.008 207 KRKQrRRKGR 0.006 205 KKKRkQRRRK 0.006 216 RPEDlEFTDM 0.006 249 EKRPvGKGRK 0.006 294 TLVLlLLVLL 0.006 305 VFLLlVFYTI 0.006 82 NFNWrFVFRF 0.006 297 LLLLvLLTVF 0.006 199 QEAQaGKKKR 0.006 295 LVLLlLVLLT 0.006 154 GPELcSVQLA 0.006 151 GARGpELCSV 0.006 9 DVPApPPVDI 0.006 298 LLLVlLTVFL 0.006 248 AEKRpVGKGR 0.006 229 VYILtGKVEA 0.006 67 KQETdVHFNS 0.005 123 VLQVwDYDRI 0.004 93 YLPTeREVSV 0.004 283 FVFFiWRRYW 0.004 203 AGKKkRKQRR 0.004 231 ILTGkVEAEF 0.004 108 PFALeEAEFR 0.004 140 SLELqLPDMV 0.004 313 TIPGqISQVI 0.004 155 PELCsVQLAR 0.004 38 VVLDdENPLT 0.003 275 WFVNpLKTFV 0.003 270 KTSFnWFVNP 0.003 54 IYVKsWVKGL 0.003 131 RISAnDFLGS 0.002

TABLE XVI 158P3D2v.1-A24-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 290 RYWRTLVLL 400.000 86 RFVFRFDYL 72.000 272 SFNWFVNPL 36.000 275 WFVNPLKTF 15.000 235 KVEAEFELL 14.400 318 ISQVIFRPL 10.080 301 VLLTVFLLL 10.080 92 DYLPTEREV 9.900 180 RGWWPVVKL 8.800 38 VVLDDENPL 8.640 255 KGRKQPEPL 8.000 25 SYELRVVIW 7.500 299 LLVLLTVFL 7.200 294 TLVLLLLVL 7.200 295 LVLLLLVLL 7.200 133 SANDFLGSL 7.200 310 VFYTIPGQI 7.000 171 CNLFRCRRL 6.000 32 IWNTEDVVL 6.000 154 GPELCSVQL 6.000 311 FYTIPGQIS 6.000 300 LVLLTVFLL 6.000 268 RPKTSFNWF 5.760 69 ETDVHFNSL 5.760 214 KGRPEDLEF 5.280 137 FLGSLELQL 4.800 291 YWRTLVLLL 4.800 55 YVKSWVKGL 4.000 224 DMGGNVYIL 4.000 165 NGAGPRCNL 4.000 287 IWRRYWRTL 4.000 265 KPSRPKTSF 4.000 303 LTVFLLLVF 3.600 298 LLLVLLTVF 3.600 278 NPLKTFVFF 3.600 232 LTGKVEAEF 3.080 277 VNPLKTFVF 3.000 75 NSLTGEGNF 3.000 166 GAGPRCNLF 2.880 306 FLLLVFYTI 2.520 83 FNWRFVFRF 2.000 101 SVWRRSGPF 2.000 81 GNFNWRFVF 2.000 314 IPGQISQVI 1.680 124 LQVWDYDRI 1.500 24 ISYELRVVI 1.440 10 VPAPPPVDI 1.200 46 LTGEMSSDI 1.200 108 PFALEEAEF 1.100 305 VFLLLVFYT 0.900 54 IYVKSWVKG 0.825 289 RRYWRTLVL 0.800 212 RRKGRPEDL 0.800 234 GKVEAEFEL 0.792 229 VYILTGKVE 0.750 140 SLELCLPDM 0.750 116 FRQPAVLVL 0.720 128 DYDRISAND 0.700 221 EFTDMGGNV 0.600 130 DRISANDFL 0.600 292 WRTLVLLLL 0.560 115 EFRQPAVLV 0.500 88 VFRFDYLPT 0.500 284 VFFIWRRYW 0.500 149 VRGARGPEL 0.440 135 NDFLGSLEL 0.440 114 AEFRCPAVL 0.400 103 WRRSGPFAL 0.400 66 DKQEIDVHF 0.360 293 RTLVLLLLV 0.360 315 PGQISQVIF 0.300 67 KQETDVHFN 0.300 190 EAEDVEREA 0.277 129 YDRISANDF 0.240 175 RCRRLRGWW 0.240 259 QPEPLEKPS 0.216 276 FVNPLKTFV 0.216 297 LLLLVLLTV 0.210 150 RGARGPELC 0.200 270 KTSFNWFVN 0.200 164 RNGAGPRCN 0.200 79 GEGNFNWRF 0.200 178 RLRGWWPVV 0.200 193 DVEREAQEA 0.198 62 GLEHDKQET 0.198 50 MSSDIYVKS 0.185 16 VDIKPRQPI 0.180 296 VLLLLVLLT 0.180 159 SVQLARNGA 0.180 144 QLPDMVRGA 0.180 106 SGPFALEEA 0.165 183 WPVVKLKEA 0.165 230 YILTGKVEA 0.165 80 EGNFNWRFV 0.150 237 EAEFELLTV 0.150 43 ENPLTGEMS 0.150 44 NPLTGEMSS 0.150 30 VVIWNTEDV 0.150 223 TDMGGNVYI 0.150 22 QPISYELRV 0.150

TABLE XVII 158P3D2v.1-A24-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 290 RYWRtLVLLL 480.000 54 IYVKsWVKGL 300.000 128 DYDRiSANDF 120.000 136 DFLGsLELQL 36.000 115 EFRQpAVLVL 20.000 82 NFNWrFVFRF 15.000 153 RGPElCSVQL 14.400 293 RTLVlLLLVL 14.400 305 VFLLlVFYTI 12.600 19 KPRQpISYEL 12.320 170 RCNLfRCRRL 12.000 25 SYELrVVIWN 10.500 300 LVLLtVFLLL 10.080 92 DYLPtEREVS 9.000 229 VYILtGKVEA 8.250 164 RNGAgPRCNL 8.000 37 DVVLdDENPL 7.200 294 TLVLlLLVLL 7.200 298 LLLVlLTVFL 7.200 317 QISQvIFRPL 6.720 299 LLVLlTVFLL 6.000 113 EAEFrQPAVL 6.000 291 YWRTlVLLLL 5.600 271 TSFNwFVNPL 4.800 134 ANDFlGSLEL 4.400 233 TGKVeAEFEL 4.400 148 MVRGaRGPEL 4.400 31 VIWNtEDVVL 4.000 286 FIWRrYWRTL 4.000 132 ISANdFLGSL 4.000 102 VWRRsGPFAL 4.000 277 VNPLkTFVFF 3.600 276 FVNPlKTFVF 3.600 297 LLLLvLLTVF 3.600 231 ILTGkVEAEF 3.080 80 EGNFnWRFVF 3.000 78 TGEGnFNWRF 3.000 100 VSVWrRSGPF 3.000 313 TIPGqISQVI 2.520 302 LLTVfLLLVF 2.400 165 NGAGpRCNLF 2.400 107 GPFAlEEAEF 2.200 216 RPEDlEFTDM 2.160 314 IPGQiSQVIF 2.000 74 FNSLtGEGNF 2.000 274 NWFVnPLKTF 2.000 9 DVPApPPVDI 1.500 278 NPLKtFVFFI 1.500 123 VLQVwDYDRI 1.500 309 LVFYtIPGQI 1.400 282 TFVFfIWRRY 1.050 222 FTDMgGNVYI 1.000 275 WFVNpLKTFV 0.900 139 GSLElQLPDM 0.900 234 GKVEaEFELL 0.864 239 EFELlTVEEA 0.825 211 RRRKgRPEDL 0.800 289 RRYWrTLVLL 0.800 285 FFIWrRYWRT 0.750 73 HFNSlTGEGN 0.750 221 EFTDmGGNVY 0.720 68 QETDvHFNSL 0.691 223 TDMGgNVYIL 0.600 310 VFYTiPGQIS 0.600 311 FYTIpGQISQ 0.500 173 LFRCrRLRGW 0.500 85 WRFVfRFDYL 0.480 61 KGLEhDKQET 0.475 213 RKGRpEDLEF 0.440 179 LRGWwPVVKL 0.440 267 SRPKtSFNWF 0.432 258 KQPEpLEKPS 0.432 67 KQETdVHFNS 0.420 129 YDRIsANDFL 0.400 254 GKGRkQPEPL 0.400 288 WRRYwRTLVL 0.400 117 RQPAvLVLQV 0.360 29 RVVIwNTEDV 0.300 235 KVEAeFELLT 0.300 264 EKPSrPKTSF 0.300 21 RQPIsYELRV 0.300 105 RSGPfALEEA 0.264 214 KGRPeDLEFT 0.240 131 RISAnDFLGS 0.240 1 MWIDiFPQDV 0.216 296 VLLLlVLLTV 0.210 268 RPKTsFNWFV 0.200 265 KPSRpKTSFN 0.200 65 HDKQeTDVHF 0.200 150 RGARgPELCS 0.200 227 GNVYiLTGKV 0.198 154 GPELcSVQLA 0.180 303 LTVFlLLVFY 0.180 38 VVLDdENPLT 0.180 312 YTIPgQISQV 0.180 158 CSVQlARNGA 0.180 143 LQLPdMVRGA 0.180 295 LVLLlLVLLT 0.180 244 TVEEaEKRPV 0.180 75 NSLTgEGNFN 0.180

TABLE XVIII 158P3D2v.1-B7-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 255 KGRKQPEPL 40.000 154 GPELCSVQL 24.000 300 LVLLTVFLL 20.000 55 YVKSWVKGL 20.000 168 GPRCNLFRC 20.000 295 LVLLLLVLL 20.000 38 VVLDDENPL 20.000 133 SANDFLGSL 12.000 10 VPAPPPVDI 12.000 165 NGAGPRCNL 9.000 314 IPGQISQVI 8.000 180 RGWWPVVKL 6.000 235 KVEAEFELL 6.000 294 TLVLLLLVL 4.000 94 LPTEREVSV 4.000 22 QPISYELRV 4.000 301 VLLTVFLLL 4.000 291 YWRTLVLLL 4.000 318 ISQVIFRPL 4.000 103 WRRSGPFAL 4.000 299 LLVLLTVFL 4.000 118 QPAVLVLQV 4.000 137 FLGSLELQL 4.000 287 IWRRYWRTL 4.000 171 CNLFRCRRL 4.000 224 DMGGNVYIL 4.000 19 KPRQPISYE 3.000 178 RLRGWWPVV 2.000 183 WPVVKLKEA 2.000 114 AEFRQPAVL 1.200 69 ETDVHFNSL 1.200 276 FVNPLKTFV 1.000 27 ELRVVIWNT 1.000 30 VVIWNTEDV 1.000 228 NVYILTGKV 1.000 151 GARGPELCS 0.900 159 SVQLARNGA 0.750 148 MVRGARGPE 0.750 24 ISYELRVVI 0.600 265 KPSRPKTSF 0.600 12 APPPVDIKP 0.600 292 WRTLVLLLL 0.400 32 IWNTEDVVL 0.400 289 RRYWRTLVL 0.400 149 VRGARGPEL 0.400 46 LTGEMSSDI 0.400 306 FLLLVFYTI 0.400 272 SFNWFVNPL 0.400 234 GKVEAEFEL 0.400 278 NPLKTFVFF 0.400 130 DRISANDFL 0.400 86 RFVFRFDYL 0.400 135 NDFLGSLEL 0.400 44 NPLTGEMSS 0.400 212 RRKGRPEDL 0.400 268 RPKTSFNWF 0.400 290 RYWRTLVLL 0.400 116 FRQPAVLVL 0.400 124 LQVWDYDRI 0.400 140 SLELQLPDM 0.300 288 WRRYWRTLV 0.300 162 LARNGAGPR 0.300 115 EFRQPAVLV 0.300 175 RCRRLRGWW 0.300 214 KGRPEDLEF 0.200 80 EGNFNWRFV 0.200 302 LLTVFLLLV 0.200 297 LLLLVLLTV 0.200 261 EPLEKPSRP 0.200 107 GPFALEEAE 0.200 31 VIWNTEDVV 0.200 313 TIPGQISQV 0.200 251 RPVGKGRKQ 0.200 293 RTLVLLLLV 0.200 6 FPQDVPAPP 0.200 237 EAEFELLTV 0.180 113 EAEFRQPAV 0.180 193 DVEREAQEA 0.150 120 AVLVLQVWD 0.150 259 QPEPLEKPS 0.120 223 TDMGGNVYI 0.120 283 FVFFIWRRY 0.100 106 SGPFALEEA 0.100 101 SVWRRSGPF 0.100 150 RGARGPELC 0.100 304 TVFLLLVFY 0.100 42 DENPLTGEM 0.100 296 VLLLLVLLT 0.100 125 QVWDYDRIS 0.100 225 MGGNVYILT 0.100 144 QLPDMVRGA 0.100 102 VWRRSGPFA 0.100 4 DIFPQDVPA 0.100 88 VERFDYLPT 0.100 209 KQRRRKGRP 0.100 286 FIWRRYWRT 0.100 230 YILTGKVEA 0.100 16 VDIKPRQPI 0.090 145 LPDMVRGAR 0.090 190 EAEDVEREA 0.090

TABLE XIX 158P3D2v.1-B7-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 19 KPRQpISYEL 800.000 148 MVRGaRGPEL 200.000 37 DVVLdDENPL 20.000 300 LVLLtVFLLL 20.000 164 RNGAgPRCNL 9.000 278 NPLKtFVFFI 8.000 151 GARGpELCSV 6.000 216 RPEDlEFTDM 6.000 31 VIWNtEDVVL 4.000 298 LLLVlLTVFL 4.000 294 TLVLlLLVLL 4.000 129 YDRIsANDFL 4.000 132 ISANdFLGSL 4.000 288 WRRYwRTLVL 4.000 170 RCNLfRCRRL 4.000 22 QPISyELRVV 4.000 115 EFRQpAVLVL 4.000 153 RGPElCSVQL 4.000 293 RTLVlLLLVL 4.000 291 YWRTlVLLLL 4.000 286 FIWRrYWRTL 4.000 271 TSFNwFVNPL 4.000 233 TGKVeAEFEL 4.000 268 RPKTsFNWFV 4.000 299 LLVLlTVELL 4.000 211 RRRKgRPEDL 4.000 102 VWRRsGPFAL 4.000 317 QISQvIFRPL 4.000 134 ANDFlGSLEL 3.600 113 EAEFrQPAVL 3.600 9 DVPApPPVDI 3.000 162 LARNgAGPRC 3.000 309 LVFYtIPGQI 2.000 168 GPRCnLFRCR 2.000 223 TDMGgNVYIL 1.200 30 VVIWnTEDVV 1.000 29 RVVIwNTEDV 1.000 214 KGRPeDLEFT 1.000 185 VVKLkEAEDV 1.000 139 GSLElQLPDM 1.000 125 QVWDyDRISA 0.750 12 APPPvDIKPR 0.600 154 GPELcSVQLA 0.600 179 LRGWwPVVKL 0.600 295 LVLLlLVLLT 0.500 38 VVLDdENPLT 0.500 87 FVFRfDYLPT 0.500 101 SVWRrSGPFA 0.500 304 TVFLlLVFYT 0.500 54 IYVKsWVKGL 0.400 313 TIPGqISQVI 0.400 289 RRYWrTLVLL 0.400 136 DFLGsLELQL 0.400 234 GKVEaEFELL 0.400 254 GKGRkQPEPL 0.400 118 QPAVlVLQVW 0.400 314 IPGQiSQVIF 0.400 68 QETDvHFNSL 0.400 107 GPFAlEEAEF 0.400 123 VLQVwDYDRI 0.400 290 RYWRtLVLLL 0.400 94 LPTErEVSVW 0.400 265 KPSRpKTSFN 0.400 85 WRFVfRFDYL 0.400 261 EPLEkPSRPK 0.300 10 VPAPpPVDIK 0.300 120 AVLVlQVWDY 0.300 167 AGPRcNLFRC 0.300 287 IWRRyWRTLV 0.300 244 TVEEaEKRPV 0.300 251 RPVGkGRKQP 0.300 6 FPQDvPAPPP 0.300 296 VLLLlVLLTV 0.200 117 RQPAvLVLQV 0.200 44 NPLTgEMSSD 0.200 176 CRRLrGWWPV 0.200 183 WPVVkLKEAE 0.200 301 VLLTvFLLLV 0.200 227 GNVYiLTGKV 0.200 21 RQPIsYELRV 0.200 312 YTIPgQISQV 0.200 93 YLPTeREVSV 0.200 235 KVEAeFELLT 0.150 158 CSVQlARNGA 0.150 283 FVFFIWRRYW 0.150 255 KGRKqPEPLE 0.150 15 PVDIkPRQPI 0.135 222 FTDMgGNVYI 0.120 209 KQRRrKGRPE 0.100 105 RSGPfALEEA 0.100 27 ELRVvIWNTE 0.100 273 FNWFvNPLKT 0.100 143 LQLPdMVRGA 0.100 175 RCRRlRGWWP 0.100 276 FVNPlKTFVF 0.100 61 KGLEhDKQET 0.100 224 DMGGnVYILT 0.100 178 RLRGwWPVVK 0.100 194 VEREaQEAQA 0.100 114 AEFRqPAVLV 0.090

TABLE XX 158P3D2v.1-B3501-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 268 RPKTSENWF 120.000 265 KPSRPKTSF 40.000 278 NPLKTFVFF 20.000 214 KGRPEDLEF 9.000 314 IPGQISQVI 8.000 94 LPTEREVSV 8.000 10 VPAPPPVDI 8.000 154 GPELCSVQL 6.000 168 GPRCNLFRC 6.000 255 KGRKQPEPL 6.000 133 SANDFLGSL 6.000 318 ISQVIFRPL 5.000 75 NSLTGEGNF 5.000 118 QPAVLVLQV 4.000 24 ISYELRVVI 4.000 22 QPISYELRV 4.000 38 VVLDDENPL 3.000 55 YVKSWVKGL 3.000 175 RCRRLRGWW 3.000 166 GAGPRCNLF 3.000 180 RGWWPVVKL 2.000 183 WPVVKLKEA 2.000 283 FVFFIWRRY 2.000 304 TVFLLLVFY 2.000 121 VLVLQVWDY 2.000 44 NPLTGEMSS 2.000 19 KPRQPISYE 1.200 178 RLRGWWPVV 1.200 299 LLVLLTVFL 1.000 165 NGAGPRCNL 1.000 224 DMGGNVYIL 1.000 277 VNPLKTFVF 1.000 298 LLLVLLTVF 1.000 294 TLVLLLLVL 1.000 137 FLGSLELQL 1.000 171 CNLFRCRRL 1.000 101 SVWRRSGPF 1.000 81 GNFNWRFVF 1.000 300 LVLLTVFLL 1.000 50 MSSDIYVKS 1.000 83 FNWRFVFRF 1.000 232 LTGKVEAEF 1.000 303 LTVFLLLVF 1.000 301 VLLTVFLLL 1.000 295 LVLLLLVLL 1.000 77 LTGEGNFNW 1.000 235 KVEAEFELL 0.900 151 GARGPELCS 0.900 46 LTGEMSSDI 0.800 51 SSDIYVKSW 0.750 132 ISANDFLGS 0.750 222 FTDMGGNVY 0.600 47 TGEMSSDIY 0.600 259 QPEPLEKPS 0.600 140 SLELQLPDM 0.600 212 RRKGRPEDL 0.600 124 LQVWDYDRI 0.600 293 RTLVLLLLV 0.400 306 FLLLVFYTI 0.400 251 RPVGKGRKQ 0.400 6 FPQDVPAPP 0.400 261 EPLEKPSRP 0.400 129 YDRISANDF 0.300 291 YWRTLVLLL 0.300 17 DIKPRQPIS 0.300 27 ELRVVIWNT 0.300 287 IWRRYWRTL 0.300 69 ETDVHFNSL 0.300 103 WRRSGPFAL 0.300 237 EAEFELLTV 0.270 216 RPEDLEFTD 0.240 164 RNGAGPRCN 0.200 234 GKVEAEFEL 0.200 30 VVIWNTEDV 0.200 313 TIPGQISQV 0.200 18 IKPRQPISY 0.200 150 RGARGPELC 0.200 297 LLLLVLLTV 0.200 42 DENPLTGEM 0.200 107 GPFALEEAE 0.200 290 RYWRTLVLL 0.200 302 LLTVFLLLV 0.200 12 APPPVDIKP 0.200 31 VIWNTEDVV 0.200 276 FVNPLKTFV 0.200 228 NVYILTGKV 0.200 125 QVWDYDRIS 0.200 86 RFVFRFDYL 0.200 144 QLPDMVRGA 0.200 66 DKQETDVHF 0.200 80 EGNFNWRFV 0.200 85 WRFVFRFDY 0.200 289 RRYWRTLVL 0.200 270 KTSFNWFVN 0.200 113 EAEFRQPAV 0.180 190 EAEDVEREA 0.180 76 SLTGEGNFN 0.150 266 PSRPKTSFN 0.150 32 IWNTEDVVL 0.150 119 PAVLVLQVW 0.150

TABLE XXI 158P3D2v.1-B3501-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 19 KPRQpISYEL 120.000 216 RPEDlEFTDM 72.000 94 LPTErEVSVW 30.000 107 GPFAlEEAEF 30.000 268 RPKTsFNWFV 24.000 139 GSLElQLPDM 20.000 314 IPGQiSQVIF 20.000 118 QPAVlVLQVW 10.000 278 NPLKtFVFFI 8.000 17 DIKPrQPISY 6.000 22 QPISyELRVV 6.000 271 TSFNwFVNPL 5.000 24 ISYElRVVIW 5.000 50 MSSDiYVKSW 5.000 132 ISANdFLGSL 5.000 100 VSVWrRSGPF 5.000 46 LTGEmSSDIY 4.000 153 RGPElCSVQL 4.000 265 KPSRpKTSFN 4.000 148 MVRGaRGPEL 3.000 233 TGKVeAEFEL 3.000 151 GARGpELCSV 2.700 164 RNGAgPRCNL 2.000 120 AVLVlQVWDY 2.000 293 RTLVlLLLVL 2.000 303 LTVFlLLVFY 2.000 170 RCNLfRCRRL 2.000 37 DVVLdDENPL 1.500 31 VIWNtEDVVL 1.500 298 LLLVlLTVFL 1.000 317 QISQvIFRPL 1.000 294 TLVLlLLVLL 1.000 286 FIWRrYWRTL 1.000 299 LLVLlTVFLL 1.000 300 LVLLtVFLLL 1.000 277 VNPLkTFVFF 1.000 105 RSGPfALEEA 1.000 302 LLTVfLLLVF 1.000 74 FNSLtGEGNF 1.000 231 ILTGkVEAEF 1.000 80 EGNFnWRFVF 1.000 297 LLLLvLLTVF 1.000 165 NGAGpRCNLF 1.000 276 FVNPlKTFVF 1.000 113 EAEFrQPAVL 0.900 185 VVKLkEAEDV 0.900 214 KGRPeDLEFT 0.900 162 LARNgAGPRC 0.900 75 NSLTgEGNFN 0.750 266 PSRPkTSFNW 0.750 123 VLQVwDYDRI 0.600 154 GPELcSVQLA 0.600 84 NWRFvFRFDY 0.600 211 RRRKgRPEDL 0.600 61 KGLEhDKQET 0.600 168 GPRCnLFRCR 0.600 158 CSVQlARNGA 0.500 283 FVFFiWRRYW 0.500 76 SLTGeGNFNW 0.500 9 DVPApPPVDI 0.400 261 EPLEkPSRPK 0.400 29 RVVIwNTEDV 0.400 21 RQPIsYELRV 0.400 251 RPVGkGRKQP 0.400 309 LVFYtIPGQI 0.400 6 FPQDvPAPPP 0.400 258 KQPEpLEKPS 0.400 117 RQPAvLVLQV 0.400 313 TIPGqISQVI 0.400 221 EFTDmGGNVY 0.400 213 RKGRpEDLEF 0.300 125 QVWDyDRISA 0.300 129 YDRIsANDFL 0.300 102 VWRRsGPFAL 0.300 115 EFRQpAVLVL 0.300 288 WRRYwRTLVL 0.300 134 ANDFlGSLEL 0.300 78 TGEGnFNWRF 0.300 38 VVLDdENPLT 0.300 234 GKVEaEFELL 0.300 65 HDKQeTDVHF 0.300 291 YWRTlVLLLL 0.300 12 APPPvDIKPR 0.300 131 RISAnDFLGS 0.300 44 NPLTgEMSSD 0.300 51 SSDIyVKSWV 0.300 290 RYWRtLVLLL 0.200 282 TFVFfIWRRY 0.200 183 WPVVkLKEAE 0.200 10 VPAPpPVDIK 0.200 68 QETDvHFNSL 0.200 227 GNVYiLTGKV 0.200 93 YLPTeREVSV 0.200 30 VVIWnTEDVV 0.200 296 VLLLlVLLTV 0.200 150 RGARgPELCS 0.200 301 VLLTvFLLLV 0.200 289 RRYWrTLVLL 0.200 312 YTIPgQISQV 0.200 187 KLKEaEDVER 0.180

TABLE VIII 158P3D2v.17, ORF: 65-6175, Frame +2, A1-9-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 1930 FTDMGGNVY 62.500 626 GAEGPEIPR 45.000 1553 ETELTVAVF 45.000 1576 HIDLENRFY 25.000 40 EADIGELER 25.000 217 HGDPFQVSR 25.000 1349 ELDWGSKYY 25.000 151 ELEPGEAQL 18.000 427 RAEGLPALR 18.000 197 DVELSGVMF 18.000 583 RAEPEPPQA 18.000 1474 FSEPQISRG 13.500 1202 ESEVLASYR 13.500 272 YNEYFLFEF 11.250 1755 TGEMSSDIY 11.250 1306 SLDPFLAEA 10.000 668 MIDPTVASQ 10.000 1598 EVDGYNAWR 10.000 1511 KADPYVVVS 10.000 452 LVEPYVRVS 9.000 1146 LVEPHSGRL 9.000 1121 LIELDYSGR 9.000 267 TSCPFYNEY 7.500 1011 LSDPFARVL 7.500 786 LQEVERLQR 6.750 1344 IPDPEELDW 6.250 346 ILDQPDGQF 5.000 1267 HIVPHMLRF 5.000 327 QTLPFMATR 5.000 1927 DLEFTDMGG 4.500 703 GTEGAAVEA 4.500 821 GAERRTMTR 4.500 1466 YPESEAVLF 4.500 1945 EAEFELLTV 4.500 1200 GVESEVLAS 4.500 1688 HVETRPLYH 4.500 708 AVEAQPLLG 4.500 1137 EVEPQDLAP 4.500 585 EPEPPQAQQ 4.500 235 VLEAQKLVG 4.500 1818 ALEEAEFRQ 4.500 1379 DSDGVNLIS 3.750 1955 EAEKRPVGK 3.600 1906 AQEAQAGKK 2.700 316 LTPPSPKAF 2.500 1659 SGDPEEAQA 2.500 349 QPDGQFYQR 2.500 1215 FTELVRHLT 2.250 1967 QPEPLEKPS 2.250 1786 TGEGNFNWR 2.250 734 RPEPMDGSG 2.250 745 FCLPLCHCK 2.000 485 FVELFPPLT 1.800 807 ALEVLVAGS 1.800 1186 EVEQPQVVL 1.800 1578 DLENRFYSH 1.800 1821 EAEFRQPAV 1.800 471 SAEAAAPEW 1.800 1129 RLEPSVPSE 1.800 881 LAEEPQPPL 1.800 1184 LLEVEQPQV 1.800 1848 SLELQLPDM 1.800 543 GSPPGAGLR 1.500 964 TSELPPDLL 1.350 827 MTRPNALDR 1.250 1989 KTFVFFIWR 1.250 977 AGLPSSLHR 1.250 1777 ETDVHFNSL 1.250 1853 LPDMVRGAR 1.250 512 ATHVPDLRR 1.250 2020 YTIPGQISQ 1.250 1073 FGPPVFLGR 1.250 2011 LTVFLLLVF 1.250 1097 RPELQFFPL 1.125 1540 FGEILELSI 1.125 1742 NTEDVVLDD 1.125 1874 GAGPRCNLF 1.000 368 RAGTKGFIK 1.000 1159 NVCPVLREF 1.000 2012 TVFLLLVFY 1.000 2028 QVIFRPLHK 1.000 1099 ELQFFPLRK 1.000 506 LVDAALATH 1.000 1621 RCGLPAPEY 1.000 710 EAQPLLGAR 1.000 1747 VLDDENPLT 1.000 1029 VLEQTLSPL 0.900 1057 QEEPPLVII 0.900 1896 LKEAEDVER 0.900 110 QVELDLKYQ 0.900 291 LLEITVSGV 0.900 947 KLELFLRLG 0.900 689 RLEEQLGRG 0.900 1397 EAEVKGTVS 0.900 1193 VLEVAGQGV 0.900 1943 KVEAEFELL 0.900 1311 LAEAGISRQ 0.900 1898 EAEDVEREA 0.900 1770 GLEHDKQET 0.900 1168 RVEVLFWGL 0.900

TABLE IX 158P3D2v.17, ORF: 65-6175, Frame +2, A1-10-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 452 LVEPyVRVSF 180.000 1200 GVESeVLASY 90.000 485 FVELfPPLTR 45.000 1967 QPEPlEKPSR 45.000 346 ILDQpDGQFY 25.000 43 IGELfRWPHY 22.500 1137 EVEPqDLAPL 18.000 1011 LSDPfARVLI 15.000 1474 FSEPqISRGI 13.500 972 LPEPsAGLPS 11.250 1215 FTELvRHLTV 11.250 476 APEWnEQLSF 11.250 1511 KADPyVVVSA 10.000 1626 APEYrAGAVK 9.000 807 ALEVlVAGSR 9.000 1168 RVEVlFWGLR 9.000 583 RAEPePPQAQ 9.000 1129 RLEPsVPSEV 9.000 1146 LVEPhSGRLS 9.000 1306 SLDPfLAEAG 5.000 1874 GAGPrCNLFR 5.000 1984 FVNPlKTFVF 5.000 1346 DPEElDWGSK 4.500 401 DIEKnLLLPR 4.500 708 AVEAqPLLGA 4.500 427 RAEGlPALRL 4.500 1097 RPELqFFPLR 4.500 1290 MEETgDMMPK 4.500 637 EVEVeELLPL 4.500 1943 KVEAeFELLT 4.500 1543 ILELsISLPA 4.500 197 DVELsGVMFS 4.500 947 KLELfLRLGL 4.500 1379 DSDGvNLISM 3.750 981 SSLHrDDFSY 3.750 399 CSDIeKNLLL 3.750 521 ISHPgRAAGF 3.000 1906 AQEAqAGKKK 2.700 127 WSEEdFGAPI 2.700 786 LQEVeRLQRK 2.700 1725 DIKPrQPISY 2.500 1576 HIDLeNRFYS 2.500 1747 VLDDeNPLTG 2.500 2011 LTVFlLLVEY 2.500 1117 AAFQiIELDY 2.500 1930 FTDMgGNVYI 2.500 40 EADIgELFRW 2.500 193 DDEPdVELSG 2.250 1786 TGEGnFNWRF 2.250 703 GTEGaAVEAQ 2.250 38 GPEAdIGELF 2.250 585 EPEPpQAQQG 2.250 1057 QEEPpLVIIN 2.250 1502 NLAPaDPNGK 2.000 938 GAAPgEVCAK 2.000 235 VLEAqKLVGV 1.800 1029 VLEQtLSPLW 1.800 1821 EAEFrQPAVL 1.800 626 GAEGpEIPRA 1.800 1392 IQDQgEAEVK 1.500 267 TSCPfYNEYF 1.500 1360 LQELqGQHNF 1.350 1754 LTGEmSSDIY 1.250 340 RMDLgIILDQ 1.250 1344 IPDPeELDWG 1.250 266 GTSCpFYNEY 1.250 217 HGDPfQVSRA 1.250 1659 SGDPeEAQAL 1.250 1777 ETDVhFNSLT 1.250 1572 IGEThIDLEN 1.125 629 GPEIpRAMEV 1.125 181 ENELeLELEQ 1.125 1755 TGEMsSDIYV 1.125 1466 YPESeAVLFS 1.125 1661 DPEEaQALLV 1.125 1112 AGELiAAFQL 1.125 620 HLDAsPGAEG 1.000 841 LVHSlNLLAK 1.000 1313 EAGIsRQLLK 1.000 1481 RGIPqNRPIK 1.000 949 ELFLrLGLGK 1.000 1683 QLVPeHVETR 1.000 868 VALAkKLLAK 1.000 191 DLDDePDVEL 1.000 315 GLTPpSPKAF 1.000 1159 NVCPvLREFR 1.000 1349 ELDWgSKYYA 1.000 1121 LIELdYSGRL 0.900 917 VVEEeRGRDC 0.900 1927 DLEFtDMGGN 0.900 1578 DLENrFYSHH 0.900 689 RLEEqLGRGS 0.900 725 EEELgTHAQR 0.900 1952 TVEEaEKRPV 0.900 183 ELELeLEQDL 0.900 291 LLEItVSGVG 0.900 1184 LLEVeQPQVV 0.900 471 SAEAaAPEWN 0.900 1848 SLELqLPDMV 0.900 1164 LREFrVEVLF 0.900

TABLE X 158P3D2v.16, ORF: 65-6175, Frame +2 A0201-9-mers  Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 2010 LLTVFLLLV 1033.404 2005 LLLLVLLTV 1006.209 1490 KLLVRVYVV 849.359 451 VLVEPYVRV 727.166 873 KLLAKLRFL 699.907 1010 GLSDPFARV 541.810 1994 FIWRRYWRT 440.113 571 LLLAVSMQV 437.482 658 LLFGVLFEA 416.858 2014 FLLLVFYTI 337.376 98 ALVDENLQV 285.163 445 ALHDQRVLV 285.163 839 KLLVHSLNL 276.643 2009 VLLTVFLLL 255.302 2007 LLVLLTVFL 199.738 895 WMLSGQRRV 170.990 1155 SLPPNVCPV 159.970 2008 LVLLTVFLL 156.843 1984 FVNPLKTFV 153.971 971 LLPEPSAGL 148.896 1163 VLREFRVEV 140.004 2004 VLLLLVLLT 107.808 286 RLQDLLLEI 98.381 1845 FLGSLELQL 98.267 650 VLAPCEDFL 97.872 1243 YLQPPLSIL 92.666 234 TVLEAQKLV 92.322 840 LLVHSLNLL 83.527 1332 GLLNQGPGL 79.041 435 RLGLLGSLV 69.552 86 QLQNAGHLV 69.552 103 NLQVSPIQV 69.552 1710 WIDIFPQDV 66.867 781 RLDQGLQEV 63.988 1171 VLFWGLRGL 61.810 2 ALTVSVQRL 49.134 510 ALATHVPDL 49.134 79 TLVISLQQL 49.134 874 LLAKLRFLA 48.984 1041 LLVFEQLIV 48.478 1746 VVLDDENPL 48.205 437 GLLGSLVRA 42.278 1003 VLAADDSGL 36.316 847 LLAKQGLRL 36.316 572 LLAVSMQVL 34.246 553 SLQGLNEGV 34.080 406 LLLPRGVPA 31.249 1635 KVGSKVFLT 30.444 1756 GEMSSDIYV 27.521 1739 VIWNTEDVV 27.109 2003 LVLLLLVLL 27.042 802 AQLKQALEV 26.092 1464 LIYPESEAV 25.492 1463 FLIYPESEA 22.853 2021 TIPGQISQV 21.996 499 QLRDDAPLV 21.672 1082 ALAAPRVKL 21.362 430 GLPALRLGL 21.362 1747 VLDDENPLT 20.776 1571 LIGETHIDL 20.473 2002 TLVLLLLVL 20.145 931 SLMLTAPGA 18.382 1701 GLLQGSLHM 18.382 1033 TLSPLWDEL 17.795 291 LLEITVSGV 17.405 1306 SLDPFLAEA 17.368 438 LLGSLVRAL 16.705 549 GLRDSLQGL 15.310 1531 YIPKQLNPI 15.177 225 RAQDFQVGV 15.050 643 LLPLPENVL 14.890 498 LQLRDDAPL 13.624 1018 VLISTQCQT 12.668 1244 LQPPLSILV 11.988 63 VQVVNCSRV 11.988 1224 VVFKDTAPL 11.757 1938 YILTGKVEA 11.626 60 CLSVQVVNC 11.426 335 RIGTFRMDL 11.162 1040 ELLVFEQLI 11.001 184 LELELEQDL 10.712 328 TLPFMATRI 10.433 1703 LQGSLHMWI 9.890 1055 HLQEEPPLV 9.696 215 LAHGDPFQV 9.525 642 ELLPLPENV 9.457 1852 QLPDMVRGA 9.370 1261 TVLVGSHIV 9.232 1113 GELIAAFQL 8.914 1614 ILAGLCQRC 8.446 1639 KVFLTPPET 8.444 2001 RTLVLLLLV 8.221 1193 VLEVAGQGV 7.567 1184 LLEVEQPQV 7.567 1683 QLVPEHVET 7.452 654 CEDFLLFGV 7.216 1557 TVAVFDHDL 7.103 638 VEVEELLPL 6.659 867 KVALAKKLL 6.542 888 PLPDVLVWM 5.669

TABLE XI 158P3D2v.17, ORF: 65-6175, Frame +2, A0201-10-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 290 LLLEiTVSGV 4125.456 2009 VLLTvFLLLV 3823.593 276 FLFEfHDTRL 1490.711 657 FLLFgVLFEA 1127.969 2004 VLLLlVLLTV 1006.209 2006 LLLVlLTVFL 739.032 839 KLLVhSLNLL 636.279 2007 LLVLlTVFLL 484.457 214 ALAHgDPFQV 403.402 880 FLAEePQPPL 402.895 873 KLLAkLRFLA 373.146 1243 YLQPpLSILV 319.939 1801 YLPTeREVSV 319.939 461 FLGQeGETSV 319.939 570 RLLLaVSMQV 257.342 650 VLAPcEDFLL 210.633 1319 QLLKpPLKKL 181.794 2012 TVFLlLVFYT 177.011 1178 GLGRvHLLEV 159.970 1192 VVLEvAGQGV 156.947 1463 FLIYpESEAV 156.770 571 LLLAvSMQVL 126.710 1702 LLQGsLHMWI 97.547 846 NLLAkQGLRL 79.041 1986 NPLKtFVFFI 70.254 1549 SLPAeTELTV 69.552 1028 RVLEsITLSPL 65.219 241 LVGVnINPYV 56.902 869 ALAKkLLAKL 49.134 2002 TLVLlLLVLL 49.134 810 VLVAgSRQFC 46.451 498 LQLRdDAPLV 44.356 658 LLFGvLFEAT 43.639 556 GLNEgVGQGI 42.774 1734 YELRvVIWNT 42.542 65 VVNCsRVFSL 42.390 1994 FIWRrYWRTL 38.130 437 GLLGsLVRAL 36.359 1183 HLLEvEQPQV 35.874 760 SCWEdHTWRL 35.591 222 QVSRaQDFQV 35.298 450 RVLVePYVRV 33.776 888 PLPDvLVWML 33.239 157 AQLErRAVAL 32.857 1010 GLSDpFARVL 27.292 777 KVAErLDQGL 26.823 85 QQLQnAGHLV 26.092 564 GIWFrGRLLL 24.380 203 VMFSpLKSRA 22.569 2008 LVLLtVFLLL 22.339 1944 VEAEfELLTV 21.680 497 RLQLrDDAPL 21.362 844 SLNLlAKQGL 21.362 1809 SVWRrSGPFA 19.844 991 FQLRaHLYQA 19.718 1491 LLVRvYVVKA 19.425 649 NVLApCEDFL 18.639 405 NLLLpRGVPA 18.382 1455 LVGKfKGSFL 17.477 235 VLEAqKLVGV 17.405 1739 VIWNtEDVVL 16.993 1746 VVLDdENPLT 16.816 847 LLAKqGLRLL 16.705 1795 FVFRfDYLPT 16.647 1535 QLNPiFGEIL 16.308 1825 RQPAvLVLQV 16.219 424 RLYRaEGLPA 15.898 1305 KSLDpFLAEA 15.049 1833 QVWDyDRISA 14.793 1668 LLVLrRWQEM 14.358 286 RLQDlLLEIT 14.118 295 TVSGvGVTSV 13.997 12 GLTGtHDRQV 13.910 352 GQFYqRWVPL 13.624 1831 VLQVwDYDRI 13.036 1018 VLIStQCQTT 12.668 1639 KVFLtPPETL 11.861 530 FNPTfGPAWV 11.487 1115 LIAAfQLIEL 11.485 1082 ALAApRVKLM 11.426 1033 TLSPlWDELL 10.468 908 IPAQdVLFSV 10.296 2020 YTIPgQISQV 10.220 1427 FEDW1NVFPL 10.196 1047 LIVDgRREHL 10.032 245 NINPyVAVQV 9.563 143 IIPNvGFQEL 9.488 2003 LVLLlLVLLT 9.433 1771 LEHDkQETDV 9.426 243 GVNInPYVAV 9.129 453 VEPYvRVSFL 8.933 175 GQQDdEENEL 8.880 1155 SLPPnVCPVL 8.759 86 QLQNaGHLVL 8.759 1676 EMPGfGIQLV 8.665 832 ALDRcRGKLL 8.545 643 LLPLpENVLA 8.446 840 LLVHsLNLLA 8.446 1040 ELLVfEQLIV 7.913 72 FSLRpLGTLV 7.727

TABLE XII 158P3D2v.17, ORF: 65-6175, Frame +2, A3-9-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 9 amino acids; and the end position for each peptide is the start position plus eight.i; Pos Subsequence Score 1319 QLLKPPLKK 90.000 276 FLFEFHDTR 90.000 1089 KLMEDPYQR 81.000 869 ALAKKLLAK 60.000 858 GLRRRNVQK 60.000 1829 VLVLQVWDY 54.000 1989 KTFVFFIWR 54.000 1491 LLVRVYVVK 45.000 1099 ELQFFPLRK 36.000 1831 VLQVWDYDR 36.000 1757 EMSSDIYVK 27.000 1430 WLNVFPLYR 24.000 203 VMFSPLKSR 22.500 304 VLQRRGDEK 20.000 953 RLGLGKQAK 20.000 1950 LLTVEEAEK 20.000 23 LTFRGFTQK 15.000 2014 FLLLVFYTI 12.150 982 SLHRDDFSY 12.000 1310 FLAEAGISR 12.000 658 LLFGVLFEA 10.125 599 RLTRKKKKK 10.000 1761 DIYVKSWVK 9.000 1706 SLHMWIDIF 9.000 1419 SLKEEFNHF 9.000 1490 KLLVRVYVV 8.100 2009 VLLTVFLLL 8.100 2028 QVIFRPLHK 6.000 1064 IINVFDHNK 6.000 232 GVTVLEAQK 6.000 1482 GIPQNRPIK 6.000 560 GVGQGIWFR 5.400 839 KLLVHSLNL 5.400 2006 LLLVLLTVF 4.500 768 RLQSSNCVR 4.000 852 GLRLLRGLR 3.600 1864 ELCSVQLAR 3.600 1850 ELQLPDMVR 3.600 430 GLPALRLGL 3.600 2024 GQISQVIFR 3.240 1767 WVKGLEHDK 3.000 1036 PLWDELLVF 3.000 2012 TVFLLLVFY 3.000 1399 EVKGTVSPK 2.700 1623 GLPAPEYRA 2.700 1332 GLLNQGPGL 2.700 2002 TLVLLLLVL 2.700 549 GLRDSLQGL 2.700 300 GVTSVLQRR 2.700 1010 GLSDPFARV 2.700 1932 DMGGNVYIL 2.430 1247 PLSILVIER 2.400 595 STLSRLTRK 2.250 1243 YLQPPLSIL 2.025 437 GLLGSLVRA 2.025 327 QTLPFMATR 2.025 1880 NLFRCRRLR 2.000 596 TLSRLTRKK 2.000 2 ALTVSVQRL 1.800 635 AMEVEVEEL 1.800 1701 GLLQGSLHM 1.800 2010 LLTVFLLLV 1.800 450 RVLVEPYVR 1.800 846 NLLAKQGLR 1.800 1684 LVPEHVETR 1.800 510 ALATHVPDL 1.800 286 RLQDLLLEI 1.800 864 VQKKVALAK 1.800 899 GQRRVAWAR 1.620 1676 EMPGFGIQL 1.620 1987 PLKTFVFFI 1.620 141 ELIIPNVGF 1.350 315 GLTPPSPKA 1.350 1267 HIVPHMLRF 1.350 1145 PLVEPHSGR 1.350 971 LLPEPSAGL 1.350 840 LLVHSLNLL 1.350 79 TLVISLQQL 1.350 2005 LLLLVLLTV 1.350 1306 SLDPFLAEA 1.350 1535 QLNPIFGEI 1.215 284 RLRLQDLLL 1.200 1845 FLGSLELQL 1.200 847 LLAKQGLRL 1.200 1889 GWWPVVKLK 1.013 1320 LLKPPLKKL 1.012 854 RLLRGLRRR 0.900 1526 DTKERYIPK 0.900 1029 VLEQTLSPL 0.900 643 LLPLPENVL 0.900 420 RLRVRLYRA 0.900 571 LLLAVSMQV 0.900 650 VLAPCEDFL 0.900 1033 TLSPLWDEL 0.900 939 AAPGEVCAK 0.900 337 GTFRMDLGI 0.900 1318 RQLLKPPLK 0.900 992 QLRAHLYQA 0.900 996 HLYQARGVL 0.900 331 FMATRIGTF 0.900

TABLE XIII 158P3D2v.17, ORF: 65-6175, Frame +2, A3-10-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 858 GLRRrNVQKK 180.000 22 KLTFrGFTQK 180.000 1490 KLLVrVYVVK 135.000 1886 RLRGwWPVVK 90.000 949 ELFLrLGLGK 60.000 1472 VLFSePQISR 60.000 1989 KTFVfFIWRR 40.500 240 KLVGvNINPY 40.500 785 GLQEvERLQR 36.000 1502 NLAPaDPNGK 30.000 913 VLFSvVEEER 30.000 714 LLGArPEEEK 30.000 1683 QLVPeHVETR 27.000 852 GLRLlRGLRR 24.000 1412 TLKIyNRSLK 20.000 768 RLQSsNCVRK 20.000 1895 KLKEaEDVER 18.000 596 TLSRlTRKKK 10.000 1949 ELLTvEEAEK 9.000 1063 VIINvFDHNK 9.000 1120 QLIElDYSGR 9.000 1296 MMPKgPQGQK 9.000 2007 LLVLlTVELL 8.100 826 TMTRpNALDR 8.000 435 RLGLlGSLVR 8.000 298 GVGVtSVLQR 7.200 2010 LLTVfLLLVF 6.000 510 ALAThVPDLR 6.000 863 NVQKkVALAK 6.000 407 LLPRgVPAER 6.000 1218 LVRHlTVVFK 6.000 441 SLVRaLHDQR 6.000 331 FMATrIGTFR 6.000 841 LVHSlNLLAK 6.000 982 SLHRdDFSYF 6.000 1828 AVLVlQVWDY 5.400 1830 LVLQvWDYDR 5.400 315 GLTPpSPKAF 4.500 276 FLFEfHDTRL 4.500 1939 ILTGkVEAEF 4.500 2005 LLLLvLLTVF 4.500 1454 HLVGkFKGSF 4.050 839 KLLVhSLNLL 4.050 556 GLNEgVGQGI 4.050 1950 LLTVeEAEKR 4.000 1078 FLGRaLAAPR 4.000 992 QLRAhLYQAR 4.000 346 ILDQpDGQFY 4.000 947 KLELfLRLGL 3.600 411 GVPAeRPWAR 3.600 303 SVLQrRGDEK 3.000 2009 VLLTvFLLLV 2.700 938 GAAPgEVCAK 2.700 1535 QLNPIFGEIL 2.700 650 VLAPcEDFLL 2.700 1491 LLVRvYVVKA 2.700 1318 RQLLkPPLKK 2.700 1200 GVESeVLASY 2.700 873 KLLAkLRFLA 2.700 1055 HLQEePPLVI 2.700 1163 VLREfRVEVL 2.700 1175 GLRGlGRVHL 2.700 1033 TLSPlWDELL 2.700 658 LLFGvLFEAT 2.250 202 GVMFsPLKSR 2.025 657 FLLFgVLFEA 2.025 424 RLYRaEGLPA 2.000 893 LVWMlSGQRR 2.000 846 NLLAkQGLRL 1.800 892 VLVWmLSGQR 1.800 73 SLRPlGTLVI 1.800 1852 QLPDmVRGAR 1.800 2027 SQVIfRPLHK 1.800 266 GTSCpFYNEY 1.800 1178 GLGRvHLLEV 1.800 1409 AVATlKIYNR 1.800 1702 LLQGsLHMWI 1.800 564 GIWFrGRLLL 1.800 807 ALEVlVAGSR 1.800 880 FLAEePQPPL 1.350 1639 KVFLtPPETL 1.350 571 LLLAvSMQVL 1.350 290 LLLEiTVSGV 1.350 1399 EVKGtVSPKK 1.350 45 ELFRwPHYGA 1.350 1701 GLLQgSLHMW 1.350 1718 VPAPpPVDIK 1.350 1155 SLPPnVCPVL 1.350 2004 VLLLlVLLTV 1.350 2002 TLVLlLLVLL 1.350 437 GLLGsLVRAL 1.215 1756 GEMSsDIYVK 1.215 1617 GLCQrCGLPA 1.200 1869 QLARnGAGPR 1.200 485 FVELfPPLTR 1.200 86 QLQNaGHLVL 1.200 517 DLRRiSHPGR 1.200 1319 QLLKpPLKKL 1.012 1224 VVFKdTAPLF 1.000 13 LTGThDRQVK 1.000

TABLE XIV 158P3D2v.17, ORF: 65-6175, Frame +2, A1101-9-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 2028 QVIFRPLHK 6.000 232 GVTVLEAQK 6.000 450 RVLVEPYVR 3.600 1318 RQLLKPPLK 2.700 560 GVGQGIWFR 2.400 1989 KTFVFFIWR 2.400 23 LTFRGFTQK 2.000 1767 WVKGLEHDK 2.000 368 RAGTKGFIK 1.800 595 STLSRLTRK 1.500 953 RLGLGKQAK 1.200 858 GLRRRNVQK 1.200 864 VQKKVALAK 1.200 1319 QLLKPPLKK 1.200 300 GVTSVLQRR 1.200 1482 GIPQNRPIK 1.200 2024 GQISQVIFR 1.080 1089 KLMEDPYQR 0.960 869 ALAKKLLAK 0.800 893 LVWMLSGQR 0.800 816 RQFCHGAER 0.720 899 GQRRVAWAR 0.720 769 LQSSNCVRK 0.600 1399 EVKGTVSPK 0.600 599 RLTRKKKKK 0.600 950 LFLRLGLGK 0.600 797 GPGACAQLK 0.600 607 KARRDQTPK 0.600 1906 AQEAQAGKK 0.600 1526 DTKERYIPK 0.600 1491 LLVRVYVVK 0.600 1761 DIYVKSWVK 0.480 512 ATHVPDLRR 0.400 1684 LVPEHVETR 0.400 827 MTRPNALDR 0.400 304 VLQRRGDEK 0.400 1950 LLTVEEAEK 0.400 62 SVQVVNCSR 0.400 1064 IINVFDHNK 0.400 251 AVQVGGQRR 0.400 442 LVRALHDQR 0.400 1729 RQPISYELR 0.360 327 QTLPFMATR 0.300 745 FCLPLCHCK 0.300 1951 LTVEEAEKR 0.300 1909 AQAGKKKRK 0.300 1757 EMSSDIYVK 0.240 786 LQEVERLQR 0.240 591 AQQGSTLSR 0.240 1099 ELQFFPLRK 0.240 852 GLRLLRGLR 0.240 1665 AQALLVLRR 0.240 768 RLQSSNCVR 0.240 821 GAERRTMTR 0.240 626 GAEGPEIPR 0.240 939 AAPGEVCAK 0.200 1297 MPKGPQGQK 0.200 1503 LAPADPNGK 0.200 1935 GNVYILTGK 0.180 1430 WLNVFPLYR 0.160 1831 VLQVWDYDR 0.160 1310 FLAEAGISR 0.160 276 FLFEFHDTR 0.160 26 RGFTQKTRK 0.120 1965 RKQPEPLEK 0.120 337 GTFRMDLGI 0.120 376 KVTLSVRAR 0.120 1798 RFDYLPTER 0.120 1168 RVEVLFWGL 0.120 427 RAEGLPALR 0.120 1878 RCNLFRCRR 0.120 243 GVNINPYVA 0.120 902 RVAWARIPA 0.120 1598 EVDGYNAWR 0.120 945 CAKLELFLR 0.120 164 VALGRRLAR 0.120 1990 TFVFFIWRR 0.120 1993 FFIWRRYWR 0.120 846 NLLAKQGLR 0.120 1613 QILAGLCQR 0.120 661 GVLFEATMI 0.090 1081 RALAAPRVK 0.090 2008 LVLLTVFLL 0.090 1062 LVIINVFDH 0.090 2001 RTLVLLLLV 0.090 787 QEVERLQRK 0.090 1121 LIELDYSGR 0.080 203 VMFSPLKSR 0.080 760 SCWEDHTWR 0.080 412 VPAERPWAR 0.080 1410 VATLKIYNR 0.080 1473 LFSEPQISR 0.080 1212 SPNFTELVR 0.080 349 QPDGQFYQR 0.080 374 FIKVTLSVR 0.080 1981 FNWFVNPLK 0.080 1958 KRPVGKGRK 0.060 1314 AGISRQLLK 0.060 1889 GWWPVVKLK 0.060 1087 RVKLMEDPY 0.060

TABLE XV 158P3D2v.17, ORF: 65-6175, Frame +2, A1101-10-mers Each peptide is a portion of SEQ ID NO: 29 each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 1318 RQLLkPPLKK 5.400 841 LVHSlNLLAK 4.000 863 NVQKkVALAK 4.000 303 SVLQrRGDEK 3.000 298 GVGVtSVLQR 2.400 411 GVPAeRPWAR 2.400 1989 KTFVfFIWRR 2.400 1218 LVRHlVVFK 2.000 2027 SQVIfRPLHK 1.800 1490 KLLVrVYVVK 1.800 1886 RLRGwWPVVK 1.200 202 GVMFsPLKSR 1.200 768 RLQSsNCVRK 1.200 1168 RVEVlFWGLR 1.200 858 GLRRrNVQKK 1.200 22 KLTFrGFTQK 1.200 1830 LVLQvWDYDR 1.200 13 LTGThDRQVK 1.000 893 LVWMlSGQRR 0.800 1409 AVATlKIYNR 0.800 163 AVALgRRLAR 0.800 485 FVELfPPLTR 0.800 816 RQFChGAERR 0.720 1756 GEMSsDIYVK 0.720 1063 VIINvFDHNK 0.600 864 VQKKvALAKK 0.600 938 GAAPgEVCAK 0.600 868 VALAkKLLAK 0.600 786 LQEVeRLQRK 0.600 1392 IQDQgEAEVK 0.600 1630 RAGAvKVGSK 0.600 1399 EVKGtVSPKK 0.600 785 GLQEvERLQR 0.480 435 RLGLlGSLVR 0.480 949 ELFLrLGLGK 0.480 852 GLRLlRGLRR 0.480 249 YVAVqVGGQR 0.400 714 LLGArPEEEK 0.400 1296 MMPKgPQGQK 0.400 574 AVSMqVLEGR 0.400 1412 TLKIyNRSLK 0.400 1159 NVCPvLREFR 0.400 1502 NLAPaDPNGK 0.400 395 APGHcSDIEK 0.400 256 GQRRvTATQR 0.360 1472 VLFSePQISR 0.320 1906 AQEAqAGKKK 0.300 107 SPIQvELDLK 0.300 1895 KLKEaEDVER 0.240 1072 KFGPpVFLGR 0.240 1874 GAGPrCNLFR 0.240 1980 SFNWfVNPLK 0.200 10 LTGLtGTHDR 0.200 492 LTRSlRLQLR 0.200 596 TLSRlTRKKK 0.200 744 YFCLpLCHCK 0.200 1519 SAGReRQDTK 0.200 1626 APEYrAGAVK 0.200 313 AAGLtPPSPK 0.200 1785 LTGEgNFNWR 0.200 1718 VPAPpPVDIK 0.200 1405 SPKKaVATLK 0.200 373 GFIKvTLSVR 0.180 1612 SQILaGLCQR 0.180 1949 ELLTvEEAEK 0.180 1904 REAQeAQAGK 0.180 1172 LFWGlRGLGR 0.160 1992 VFFIwRRYWR 0.160 913 VLFSvVEEER 0.160 826 TMTRpNALDR 0.160 595 STLSrLTRKK 0.150 1964 GRKQpEPLEK 0.120 944 VCAKlELFLR 0.120 1290 MEETgDMMPK 0.120 892 VLVWmLSGQR 0.120 87 LQNAgHLVLR 0.120 1621 RCGLpAPEYR 0.120 867 KVALaKKLLA 0.120 1494 RVYVvKATNL 0.120 1313 EAGIsRQLLK 0.120 1120 QLIElDYSGR 0.120 326 SQTLpFMATR 0.120 1639 KVFLtPPETL 0.120 1097 RPELqFFPLR 0.120 1683 QLVPeHVETR 0.120 441 SLVRaLHDQR 0.120 600 LTRKkKKKAR 0.100 1481 RGIPqNRPIK 0.090 857 RGLRrRNVQK 0.090 2001 RTLVlLLLVL 0.090 1002 GVLAaDDSGL 0.090 1737 RVVIwNTEDV 0.090 1028 RVLEqTLSPL 0.090 450 RVLVePYVRV 0.090 1251 LVIErRAFGH 0.090 1398 AEVKgTVSPK 0.090 407 LLPRgVPAER 0.080 1950 LLTVeEAEKR 0.080 510 ALAThVPDLR 0.080 331 FMATrIGTFR 0.080

TABLE XVI 158P3D2v.17, ORF: 65-6175, Frame +2, A24-9-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 1355 KYYASLQEL 528.000 1998 RYWRTLVLL 400.000 1465 IYPESEAVL 360.000 1495 VYVVKATNL 300.000 1694 LYHPHSPGL 240.000 989 SYFQLRAHL 240.000 1601 GYNAWRDAF 150.000 1794 RFVFRFDYL 72.000 484 SFVELFPPL 51.840 488 LFPPLTRSL 43.200 1980 SFNWFVNPL 36.000 277 LFEFHDTRL 30.000 1423 EFNHFEDWL 30.000 1640 VFLTPPETL 30.000 228 DFQVGVTVL 30.000 1214 NFTELVRHL 28.800 533 TFGPAWVPL 24.000 279 EFHDTRLRL 24.000 1426 HFEDWLNVF 21.600 353 QFYQRWVPL 20.000 116 KYQPPEGAT 18.000 1168 RVEVLFWGL 17.280 656 DFLLFGVLF 15.000 1983 WFVNPLKTF 15.000 1094 PYQRPELQF 15.000 162 RAVALGRRL 14.400 1097 RPELQFFPL 14.400 825 RTMTRPNAL 14.400 1943 KVEAEFELL 14.400 1107 KGPWAAGEL 13.200 1322 KPPLKKLPL 12.000 48 RWPHYGAPL 12.000 873 KLLAKLRFL 12.000 839 KLLVHSLNL 12.000 862 RNVQKKVAL 12.000 1225 VFKDTAPLF 12.000 1177 RGLGRVHLL 12.000 444 RALHDQRVL 12.000 1299 KGPQGQKSL 12.000 2026 ISQVIFRPL 10.080 2009 VLLTVFLLL 10.080 323 AFHSQTLPF 10.000 1800 DYLPTEREV 9.900 308 RGDEKAAGL 9.600 686 RAGRLEEQL 9.600 837 RGKLLVHSL 9.600 144 IPNVGFQEL 9.504 1888 RGWWPVVKL 8.800 778 VAERLDQGL 8.640 1746 VVLDDENPL 8.640 1542 EILELSISL 8.640 297 SGVGVTSVL 8.400 107 SPIQVELDL 8.400 503 DAPLVDAAL 8.400 1156 LPPNVCPVL 8.400 384 RGDLPPPML 8.000 284 RLRLQDLLL 8.000 867 KVALAKKLL 8.000 335 RIGTFRMDL 8.000 1963 KGRKQPEPL 8.000 1548 ISLPAETEL 7.920 233 VTVLEAQKL 7.920 1242 PYLQPPLSI 7.500 1596 QYEVDGYNA 7.500 997 LYQARGVLA 7.500 1733 SYELRVVIW 7.500 105 QVSPIQVEL 7.392 940 APGEVCAKL 7.392 643 LLPLPENVL 7.200 845 LNLLAKQGL 7.200 1240 EQPYLQPPL 7.200 1230 APLFHPQDL 7.200 1243 YLQPPLSIL 7.200 2007 LLVLLTVFL 7.200 1841 SANDFLGSL 7.200 840 LLVHSLNLL 7.200 1146 LVEPHSGRL 7.200 971 LLPEPSAGL 7.200 2002 TLVLLLLVL 7.200 851 QGLRLLRGL 7.200 151 ELEPGEAQL 7.200 881 LAEEPQPPL 7.200 1661 DPEEAQALL 7.200 430 GLPALRLGL 7.200 1208 SYRESPNFT 7.200 707 AAVEAQPLL 7.200 79 TLVISLQQL 7.200 2003 LVLLLLVLL 7.200 761 CWEDHTWRL 7.200 475 AAPEWNEQL 7.200 2018 VFYTIPGQI 7.000 398 HCSDIEKNL 6.720 613 TPKAVPQHL 6.720 134 APIQDSEEL 6.600 38 GPEADIGEL 6.600 1483 IPQNRPIKL 6.600 635 AMEVEVEEL 6.600 1033 TLSPLWDEL 6.336 176 QQDDEENEL 6.336 2019 FYTIPGQIS 6.000

TABLE XVII 158P3D2v.17, ORF: 65-6175, Frame +2, A24-9-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length Of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 1998 RYWRtLVLLL 480.000 1762 IYVKsWVKGL 300.000 1694 LYHPhSPGLL 240.000 1530 RYIPkQLNPI 216.000 425 LYRAeGLPAL 200.000 541 LYGSpPGAGL 200.000 271 FYNEyFLFEF 198.000 1415 IYNRsLKEEF 198.000 1465 IYPEsEAVLF 180.000 1836 DYDRiSANDF 120.000 1844 DFLGsLELQL 36.000 1242 PYLQpPLSIL 30.000 338 TFRMdLGIIL 24.000 71 VFSLrPLGTL 20.000 1823 EFRQpAVLVL 20.000 1094 PYQRpELQFF 18.000 634 RAMEvEVEEL 15.840 1790 NFNWrFVFRF 15.000 2001 RTLVLLLLVL 14.400 1861 RGPELCSVQL 14.400 75 RPLGtLVISL 14.400 947 KLELfLRLGL 14.400 839 KLLVhSLNLL 14.400 1028 RVLEqTLSPL 14.400 131 DFGApIQDSF 14.000 777 KVAErLDQGL 13.824 1081 RALAaPRVKL 13.200 2013 VELLLVEYTI 12.600 1727 KPRQpISYEL 12.320 1878 RCNLfRCRRL 12.000 427 RAEGLPALRL 12.000 497 RLQLrDDAPL 12.000 1733 SYELrVVIWN 10.500 612 QTPKaVPQHL 10.080 2008 LVLLtVFLLL 10.080 1155 SLPPnVCPVL 10.080 795 KPGPgACAQL 9.600 850 KQGLrLLRGL 9.600 416 RPWArLRVRL 9.600 175 GQQDdEENEL 9.504 143 IIPNvGFQEL 9.504 104 LQVSpIQVEL 9.240 939 AAPGeVCAKL 9.240 1800 DYLPtEREVS 9.000 1495 VYVVkATNLA 9.000 1596 QYEVdGYNAW 9.000 958 KQAKaCTSEL 8.800 429 EGLPaLRLGL 8.640 1418 RSLKeEFNHF 8.640 642 ELLPLPENVL 8.640 183 ELELeLEQDL 8.640 884 EPQPpLPDVL 8.640 1609 FWPSqILAGL 8.400 106 VSPIqVELDL 8.400 1311 LAEAgISRQL 8.400 773 NCVRkVAERL 8.400 635 AMEVeVEELL 8.400 1937 VYILtGKVEA 8.250 1494 RVYVvKATNL 8.000 1872 RNGAgPRCNL 8.000 1639 KVFLtPPETL 8.000 422 RVRLyRAEGL 8.000 479 WNEQLSFVEL 7.920 1032 QTLSpLWDEL 7.920 37 CGPEaDIGEL 7.920 831 NALDrCRGKL 7.920 1601 GYNAwRDAFW 7.500 997 LYQArGVLAA 7.500 1074 GPPVfLGRAL 7.200 1229 TAPLfHPQDL 7.200 1539 IFGEiLELSI 7.200 1047 LIVDgRREHL 7.200 548 AGLRdSLQGL 7.200 1137 EVEPqDLAPL 7.200 1535 QLNPiFGEIL 7.200 1698 HSPGLLQGSL 7.200 1302 QGQKsLDPFL 7.200 1745 DVVLdDENPL 7.200 571 LLLAvSMQVL 7.200 1 MALTvSVQRL 7.200 437 GLLGsLVRAL 7.200 157 AQLErRAVAL 7.200 2002 TLVLLLLVLL 7.200 970 DLLPePSAGL 7.200 78 GTLViSLQQL 7.200 2006 LLLVLLTVFL 7.200 19 RQVKLTFRGF 7.200 844 SLNLLAKQGL 7.200 1446 GGEEeGSGHL 7.200 2025 QISQvIFRPL 6.720 51 HYGApLAGEC 6.600 1537 NPIFgEILEL 6.600 1319 QLLKpPLKKL 6.600 133 GAPIqDSFEL 6.600 1482 GIPQnRPIKL 6.600 1483 IPQNrPIKLL 6.000 1821 EAEFrQPAVL 6.000 2007 LLVLLTVFLL 6.000 1002 GVLAaDDSGL 6.000 1556 LTVAvFDHDL 6.000

TABLE XVIII 158P3D2v.17, ORF: 65-6175, Frame +2, B7-9-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 940 APGEVCAKL 240.000 134 APIQDSFEL 240.000 1230 APLFHPQDL 240.000 774 CVRKVAERL 200.000 1148 EPHSGRLSL 120.000 269 CPFYNEYFL 80.000 107 SPIQVELDL 80.000 1699 SPGLLQGSL 80.000 1610 WPSQILAGL 80.000 431 LPALRLGLL 80.000 613 TPKAVPQHL 80.000 454 EPYVRVSFL 80.000 1483 IPQNRPIKL 80.000 144 IPNVGFQEL 80.000 1156 LPPNVCPVL 80.000 1322 KPPLKKLPL 80.000 1405 SPKKAVATL 80.000 492 LTRSLRLQL 60.000 284 RLRLQDLLL 40.000 1026 TTRVLEQTL 40.000 549 GLRDSLQGL 40.000 282 DTRLRLQDL 40.000 1963 KGRKQPEPL 40.000 166 LGRRLARSL 40.000 632 IPRAMEVEV 40.000 707 AAVEAQPLL 36.000 475 AAPEWNEQL 36.000 333 ATRIGTFRM 30.000 363 DPRDTRAGT 30.000 1082 ALAAPRVKL 27.000 1661 DPEEAQALL 24.000 1862 GPELCSVQL 24.000 38 GPEADIGEL 24.000 889 LPDVLVWML 24.000 1097 RPELQFFPL 24.000 1763 YVKSWVKGL 20.000 1876 GPRCNLFRC 20.000 867 KVALAKKLL 20.000 2008 LVLLTVFLL 20.000 2003 LVLLLLVLL 20.000 105 QVSPIQVEL 20.000 1282 DPPEEEGEM 20.000 1746 VVLDDENPL 20.000 1557 TVAVFDHDL 20.000 1224 VVFKDTAPL 20.000 1313 EAGISRQLL 18.000 1616 AGLCQRCGL 18.000 1607 DAFWPSQIL 18.000 651 LAPCEDFLL 12.000 800 ACAQLKQAL 12.000 1841 SANDFLGSL 12.000 959 QAKACTSEL 12.000 162 RAVALGRRL 12.000 870 LAKKLLAKL 12.000 53 GAPLAGECL 12.000 936 APGAAPGEV 12.000 503 DAPLVDAAL 12.000 706 GAAVEAQPL 12.000 1116 IAAFQLIEL 12.000 848 LAKQGLRLL 12.000 2 ALTVSVQRL 12.000 686 RAGRLEEQL 12.000 444 RALHDQRVL 12.000 825 RTMTRPNAL 12.000 1718 VPAPPPVDI 12.000 510 ALATHVPDL 12.000 1048 IVDGRREHL 9.000 1873 NGAGPRCNL 9.000 1504 APADPNGKA 9.000 1186 EVEQPQVVL 9.000 905 WARIPAQDV 9.000 1308 DPFLAEAGI 8.000 490 PPLTRSLRL 8.000 676 QPISFEISI 8.000 670 DPTVASQPI 8.000 1108 GPWAAGELI 8.000 1245 QPPLSILVI 8.000 2022 IPGQISQVI 8.000 1075 PPVFLGRAL 8.000 624 SPGAEGPEI 8.000 1320 LLKPPLKKL 6.000 1471 AVLFSEPQI 6.000 643 LLPLPENVL 6.000 884 EPQPPLPDV 6.000 996 HLYQARGVL 6.000 1146 LVEPHSGRL 6.000 563 QGIWFRGRL 6.000 430 GLPALRLGL 6.000 72 FSLRPLGTL 6.000 542 YGSPPGAGL 6.000 971 LLPEPSAGL 6.000 1085 APRVKLMED 6.000 504 APLVDAALA 6.000 335 RIGTFRMDL 6.000 1168 RVEVLFWGL 6.000 739 DGSGPYFCL 6.000 379 LSVRARGDL 6.000 1943 KVEAEFELL 6.000 1888 RGWWPVVKL 6.000 886 QPPLPDVLV 6.000

TABLE XIX 158P3D2v.17, ORF: 65-6175, Frame +2, B7-10 mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 1727 KPRQpISYEL 800.000 1856 MVRGaRGPEL 200.000 422 RVRLyRAEGL 200.000 1483 IPQNrPIKLL 120.000 433 ALRLgLLGSL 120.000 416 RPWArLRVRL 120.000 412 VPAErPWARL 120.000 905 WARIpAQDVL 120.000 974 EPSAgLPSSL 80.000 884 EPQPpLPDVL 80.000 75 RPLGtLVISL 80.000 1376 DPGDsDGVNL 80.000 1074 GPPVfLGRAL 80.000 1134 VPSEvEPQDL 80.000 1537 NPIFgEILEL 80.000 320 SPKAfHSQTL 80.000 489 FPPLtRSLRL 80.000 206 SPLKsRARAL 80.000 795 KPGPgACAQL 80.000 1175 GLRGlGRVHL 60.000 1633 AVKVgSKVFL 60.000 1163 VLREfRVEVL 40.000 282 DTRLrLQDLL 40.000 1316 ISRQlLKPPL 40.000 68 CSRVfSLRPL 40.000 634 RAMEvEVEEL 36.000 939 AAPGeVCAKL 36.000 474 AAAPeWNEQL 36.000 509 AALAtHVPDL 36.000 382 RARGdLPPPM 30.000 1639 KVELtPPETL 30.000 1081 RALAaPRVKL 27.000 1685 VPEHvETRPL 24.000 134 APIQdSFELI 24.000 1028 RVLEqTLSPL 20.000 777 KVAErLDQGL 20.000 649 NVLApCEDFL 20.000 65 VVNCsRVFSL 20.000 1223 TVVFkDTAPL 20.000 2008 LVLLtVFLLL 20.000 4 TVSVqRLTGL 20.000 1002 GVLAaDDSGL 20.000 1745 DVVLdDENPL 20.000 1494 RVYVvKATNL 20.000 1170 EVLFwGLRGL 20.000 747 LPLChCKPCM 20.000 1455 LVGKfKGSFL 20.000 232 GVTVlEAQKL 20.000 943 EVCAkLELFL 20.000 831 NALDrCRGKL 18.000 90 AGHLvLREAL 18.000 1615 LAGLcQRCGL 18.000 869 ALAKkLLAKL 12.000 1411 ATLKIYNRSL 12.000 133 GAPIqDSFEL 12.000 799 GACAqLKQAL 12.000 157 AQLErRAVAL 12.000 591 AQQGsTLSRL 12.000 165 ALGRrLARSL 12.000 1241 QPYLqPPLSI 12.000 706 GAAVeAQPLL 12.000 1229 TAPLfHPQDL 12.000 54 APLAgECLSV 12.000 545 PPGAgLRDSL 12.000 548 AGLRdSLQGL 12.000 945 CAKLeLFLRL 12.000 962 ACTSeLPPDL 12.000 802 AQLKqALEVL 12.000 1 MALTvSVQRL 12.000 1197 AGQGvESEVL 12.000 442 LVRAlHDQRV 10.000 568 RGRLlLAVSM 10.000 936 APGAaPGEVC 9.000 1872 RNGAgPRCNL 9.000 588 PPQAqQGSTL 8.000 1986 NPLKtFVFFI 8.000 1070 HNKFgPPVFL 6.000 429 EGLPaLRLGL 6.000 562 GQGIwFRGRL 6.000 1859 GARGpELCSV 6.000 1047 LIVDgRREHL 6.000 637 EVEVeELLPL 6.000 963 CTSElPPDLL 6.000 642 ELLPlPENVL 6.000 1137 EVEPqDLAPL 6.000 1319 QLLKpPLKKL 6.000 970 DLLPePSAGL 6.000 504 APLVdAALAT 6.000 988 FSYFqLRAHL 6.000 378 TLSVrARGDL 6.000 1085 APRVkLMEDP 6.000 880 FLAEePQPPL 6.000 195 EPDVeLSGVM 6.000 1924 RPEDlEFTDM 6.000 564 GIWFrGRLLL 6.000 427 RAEGlPALRL 5.400 1263 LVGShIVPHM 5.000 1492 LVRVyVVKAT 5.000 2001 RTLVlLLLVL 4.000 1535 QLNPiFGEIL 4.000

TABLE XX 158P3D2v.17, ORF: 65-6175, Frame +2, B3501-9-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 9 amino acids and the end position for each peptide is the start position plus eight. Pos Subsequence Score 1282 DPPEEEGEM 120.000 1976 RPKTSFNWF 120.000 736 EPMDGSGPY 80.000 1532 IPKQLNPIF 60.000 1405 SPKKAVATL 60.000 613 TPKAVPQHL 60.000 652 APCEDFLLF 60.000 1507 DPNGKADPY 40.000 1322 KPPLKKLPL 40.000 1973 KPSRPKTSF 40.000 940 APGEVCAKL 40.000 134 APIQDSFEL 30.000 1156 LPPNVCPVL 20.000 1610 WPSQILAGL 20.000 454 EPYVRVSFL 20.000 1230 APLFHPQDL 20.000 1148 EPHSGRLSL 20.000 1986 NPLKTFVFF 20.000 1699 SPGLLQGSL 20.000 107 SPIQVELDL 20.000 1269 VPHMLRFTF 20.000 431 LPALRLGLL 20.000 269 CPFYNEYFL 20.000 144 IPNVGFQEL 20.000 1483 IPQNRPIKL 20.000 418 WARLRVRLY 18.000 1594 ASQYEVDGY 15.000 363 DPRDTRAGT 12.000 632 IPRAMEVEV 12.000 1087 RVKLMEDPY 12.000 624 SPGAEGPEI 12.000 1408 KAVATLKIY 12.000 1661 DPEEAQALL 12.000 1097 RPELQFFPL 12.000 267 TSCPFYNEY 10.000 1922 KGRPEDLEF 9.000 1419 SLKEEFNHF 9.000 444 RALHDQRVL 9.000 959 QAKACTSEL 9.000 38 GPEADIGEL 9.000 848 LAKQGLRLL 9.000 870 LAKKLLAKL 9.000 1466 YPESEAVLF 9.000 1308 DPFLAEAGI 8.000 1802 LPTEREVSV 8.000 1108 GPWAAGELI 8.000 1245 QPPLSILVI 8.000 1139 EPQDLAPLV 8.000 670 DPTVASQPI 8.000 1718 VPAPPPVDI 8.000 1487 RPIKLLVRV 8.000 676 QPISFEISI 8.000 1550 LPAETELTV 8.000 2022 IPGQISQVI 8.000 1207 ASYRESPNF 7.500 1841 SANDFLGSL 6.000 889 LPDVLVWML 6.000 1876 GPRCNLFRC 6.000 686 RAGRLEEQL 6.000 333 ATRIGTFRM 6.000 213 RALAHGDPF 6.000 1035 SPLWDELLV 6.000 475 AAPEWNEQL 6.000 320 SPKAFHSQT 6.000 1111 AAGELIAAF 6.000 284 RLRLQDLLL 6.000 1862 GPELCSVQL 6.000 1963 KGRKQPEPL 6.000 526 RAAGFNPTF 6.000 837 RGKLLVHSL 6.000 448 DQRVLVEPY 6.000 162 RAVALGRRL 6.000 1083 LAAPRVKLM 6.000 549 GLRDSLQGL 6.000 707 AAVEAQPLL 6.000 1265 GSHIVPHML 5.000 1451 GSGHLVGKF 5.000 1783 NSLTGEGNF 5.000 72 FSLRPLGTL 5.000 200 LSGVMFSPL 5.000 379 LSVRARGDL 5.000 1034 LSPLWDELL 5.000 5 VSVQRLTGL 5.000 682 ISIGRAGRL 5.000 2026 ISQVIFRPL 5.000 1548 ISLPAETEL 5.000 1095 YQRPELQFF 4.500 706 GAAVEAQPL 4.500 651 LAPCEDFLL 4.500 1344 IPDPEELDW 4.500 886 QPPLPDVLV 4.000 1161 CPVLREFRV 4.000 531 NPTFGPAWV 4.000 884 EPQPPLPDV 4.000 936 APGAAPGEV 4.000 1059 EPPLVIINV 4.000 1621 RCGLPAPEY 4.000 109 IQVELDLKY 4.000 1677 MPGFGIQLV 4.000 118 QPPEGATGA 4.000

TABLE XXI 158P3D2v.17, ORF: 65-6175, Frame +2, B3501-9-mers Each peptide is a portion of SEQ ID NO: 29; each start position is Specified, the length of the peptide is 10 amino acids and the end position for each peptide is the start position plus nine. Pos Subsequence Score 1727 KPRQpISYEL 120.000 1234 HPQDLPEQPY 80.000 1487 RPIKlLVRVY 80.000 1924 RPEDLEFTDM 72.000 1134 VPSEvEPQDL 60.000 320 SPKAfHSQTL 60.000 1376 DPGDsDGVNL 60.000 75 RPLGtLVISL 40.000 412 VPAErPWARL 40.000 747 LPLChCKPCM 40.000 795 KPGPgACAQL 40.000 736 EPMDgSGPYF 40.000 416 RPWArLRVRL 40.000 382 RARGdLPPPM 36.000 1035 SPLWdELLVF 30.000 1802 LPTErEVSVW 30.000 1815 GPFAlEEAEF 30.000 1537 NPIFgEILEL 30.000 269 CPFYnEYFLF 30.000 1976 RPKTsFNWFV 24.000 974 EPSAgLPSSL 20.000 1093 DPYQrPELQF 20.000 2022 IPGQiSQVIF 20.000 1059 EPPLvIINVF 20.000 753 KPCMhVWSCW 20.000 884 EPQPpLPDVL 20.000 1483 IPQNrPIKLL 20.000 1074 GPPVfLGRAL 20.000 206 SPLKsRARAL 20.000 118 QPPEgATGAW 20.000 1847 GSLElQLPDM 20.000 979 LPSSLHRDDF 20.000 489 FPPLtRSLRL 20.000 634 RAMEvEVEEL 18.000 68 CSRVfSLRPL 15.000 1316 ISRQILKPPL 15.000 886 QPPLpDVLVW 15.000 981 SSLHrDDFSY 15.000 1418 RSLKeEFNHF 15.000 568 RGRLlLAVSM 12.000 1588 RANCgLASQY 12.000 195 EPDVeLSGVM 12.000 1826 QPAVLVLQVW 10.000 1685 VPEHvETRPL 9.000 945 CAKLeLFLRL 9.000 476 APEWnEQLSF 9.000 905 WARIpAQDVL 9.000 1593 LASQyEVDGY 9.000 1986 NPLKtFVFFI 8.000 1241 QPYLqPPLSI 8.000 134 APIQdSFELI 8.000 1725 DIKPrQPISY 6.000 263 TQRGtSCPFY 6.000 54 APLAgECLSV 6.000 1081 RALAaPRVKL 6.000 1730 QPISyELRVV 6.000 831 NALDrCRGKL 6.000 38 GPEAdIGELF 6.000 1117 AAFQlIELDY 6.000 1550 LPAEtELTVA 6.000 322 KAFHsQTLPF 6.000 332 MATRiGTFRM 6.000 422 RVRLyRAEGL 6.000 1297 MPKGpQGQKS 6.000 1163 VLREfRVEVL 6.000 1698 HSPGlLQGSL 5.000 521 ISHPgRAAGF 5.000 470 VSAEaAAPEW 5.000 740 GSGPyFCLPL 5.000 296 VSGVgVTSVL 5.000 1808 VSVWrRSGPF 5.000 267 TSCPfYNEYF 5.000 1758 MSSDiYVKSW 5.000 1840 ISANdFLGSL 5.000 1404 VSPKkAVATL 5.000 1732 ISYElRVVIW 5.000 483 LSFVeLFPPL 5.000 106 VSPIqVELDL 5.000 988 FSYFqLRAHL 5.000 1979 TSFNwFVNPL 5.000 1705 GSLHmWIDIF 5.000 1584 YSHHrANCGL 5.000 133 GAPIqDSFEL 4.500 651 LAPCeDFLLF 4.500 1206 LASYrESPNF 4.500 1648 LPPGsSSPTV 4.000 777 KVAErLDQGL 4.000 1507 DPNGkADPYV 4.000 1861 RGPElCSVQL 4.000 1754 LTGEmSSDIY 4.000 967 LPPDlLPEPS 4.000 908 IPAQdVLFSV 4.000 1028 RVLEqTLSPL 4.000 1973 KPSRpKTSFN 4.000 887 PPLPdVLVWM 4.000 240 KLVGvNINPY 4.000 1643 TPPEtLPPGS 4.000 398 HCSDiEKNLL 3.000 1110 WAAGeLIAAF 3.000 1365 GQHNfDEDEM 3.000

Tables XXII-XLIX

TABLE XXII 158P3D2v.1-A1-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 187 80GWWPVVK 1755316491 188 0GWWPVVKL 1441796 314 0VLLTVFLL 1441795 62 0KGLEHDKQ 1441794 251 0ELLTVEEA 1441793 125 0VLVLQVWD 1441792 313 00VLLTVFL 1441792 250 40ELLTVEE 262154 312 300VLLTVF 65803 230 FTDMGGNVY 36 34 NTEDVVLDD 25 47 TGEMSSDIY 25 18 IKPRQPISY 21 126 VLVLQVWDY 20 71 ETDVHFNSL 19 51 SSDIYVKSW 18 97 PTEREVSVW 18 326 YTIPGQISQ 18

TABLE XXIII 158P3D2v.1-A0201-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 187 80GWWPVVK 1755316492 188 0GWWPVVKL 1441812 314 0VLLTVFLL 1441812 313 00VLLTVFL 1441807 251 0ELLTVEEA 1441804 125 0VLVLQVWD 1441801 62 0KGLEHDKQ 1441794 250 40ELLTVEE 262150 312 300VLLTVF 65807 310 LL300VLLT 29 309 LLL300VLL 27 316 LLTVFLLLV 27 138 SANDFLGSL 25 232 DMGGNVYIL 24 307 VLLLL300V 24 308 LLLL300VL 24 315 VLLTVFLLL 24 320 FLLLVFYTI 24 327 TIPGQISQV 24 142 FLGSLELQL 23 238 YILTGKVEA 22 31 VIWNTEDVV 20 145 SLELQLPDM 20 149 QLPDMVRGA 20 157 ARGPELCSV 20 236 NVYILTGKV 20 248 F240ELLTV 20 2 WIDIFPQDV 19 30 VVIWNTEDV 19 38 VVLDDENPL 19 122 A120VLVLQ 19 239 ILTGKVEAE 19 283 SFNWFVNPL 19 287 FVNPLKTFV 19 290 PLKTFVFFI 19 311 L300VLLTV 19 23 PISYELRVV 18 118 FRQPA120V 18 121 PA120VLVL 18 302 YWRTLVLLL 18 335 VIFRPLHK3 18 39 VLDDENPLT 17 96 LPTEREVSV 17 301 RYWRTLVLL 17 4 DIFPQDVPA 16 10 VPAPPPVDI 16 24 ISYELRVVI 16 46 LTGEMSSDI 16 64 GLEHDKQET 16 140 NDFLGSLEL 16 305 TLVLLLL30 16 27 ELRVVIWNT 15 32 IWNTEDVVL 15 94 DYLPTEREV 15 126 VLVLQVWDY 15 146 LELQLPDMV 15 166 QLARNGAGP 15 170 NGAGPRCNL 15 231 TDMGGNVYI 15 246 AEF240ELL 15 253 LLTVEEAEK 15 298 IWRRYWRTL 15 321 LLLVFYTIP 15

TABLE XXIV 158P3D2v.1-A0203-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 124 20VLVLQVW 1761625411 249 240ELLTVE 1761625411 123 120VLNLQV 1381322579 186 180GWWPVV 1381322579 62 0KGLEHDKQ 1346719859 125 0VLVLQVWD 1346719859 188 0GWWPVVKL 1346719859 251 0ELLTVEEA 1346719859 313 00VLLTVFL 1346719859 314 0VLLTVFLL 1346719859 312 300VLLTVF 1315204197 187 80GWWPVVK 609485069 250 40ELLTVEE 2911585

TABLE XXV 158P3D2v.1-A3-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 124 20VLVLQVW 1761625417 249 240ELLTVE 1761625417 186 180GWWPVV 1381322589 123 120VLVLQV 1381322585 125 0VLVLQVWD 1346719876 314 0VLLTVFLL 1346719872 313 00VLLTVFL 1346719864 188 0GWWPVVKL 1346719863 251 0ELLTVEEA 1346719862 62 0KGLEHDKQ 1346719860 312 300VLLTVF 1315204206 187 80GWWPVVK 609485088 250 40ELLTVEE 2911590 334 QVIFRPLHK 31 183 RLR180GWW 23 253 LLTVEEAEK 23 55 YVKSWV60K 22 166 QLARNGAGP 22 59 WV60KGLEH 21 103 SVWRRSGPF 21 122 A120VLVLQ 21 268 RKQPEPLEK 21 318 TVFLLLVFY 21 147 ELQLPDMVR 20 273 PLEKPSRPK 20 161 ELCSVQLAR 19 195 KLKEAEDVE 19 206 AQEAQAGKK 19 177 NLFRCRRLR 18 320 FLLLVFYTI 18 9 DVPAPPPVD 17 45 PLTGEMSSD 17 53 DIYVKSWV6 17 73 DVHFNSLTG 17 126 VLVLQVWDY 17 153 MVRGARGPE 17 209 AQAGKKKRK 17 214 KKRKQRRRK 17 258 EAEKRPVGK 17 300 RRYWRTLVL 17 308 LLLL300VL 17 15 PVDIKPRQP  16 24 ISYELRVVI 16 29 RVVIWNTED 16 78 SLTGEGNFN 16 142 FLGSLELQL 16 193 VVKLKEAED 16 201 DVEREAQEA 16 207 QEAQAGKKK 16 222 KGRPEDLEF 16 236 NVYILTGKV 16 238 YILTGKVEA  16 239 ILTGKVEAE  16 261 KRPVGKGRK 16 263 PVGKGRKQP 16 294 FVFFIWRRY 16 309 LLL300VLL 16 315 VLLTVFLLL 16 327 TIPGQISQV 16

TABLE XXVI 158P3D2v.1-A26-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 187 80GWWPVVK 1755316490 314 0VLLTVFLL 1441814 188 0GWWPVVKL 1441805 313 00VLLTVFL 1441805 125 0VLVLQVWD 1441803 251 0ELLTVEEA 1441796 62 0KGLEHDKQ 1441793 250 40ELLTVEE 262145 312 300VLLTVF 65815 318 TVFLLLVFY 30 71 ETDVHFNSL 27 294 FVFFIWRRY 26 317 LTVFLLLVF 26 37 DVVLDDENP 22 73 DVHFNSLTG 21 101 EVSVWRRSG 21 135 DRISANDFL 21 232 DMGGNVYIL 21 103 SVWRRSGPF 20 38 VVLDDENPL 19 230 FTDMGGNVY 19 245 EAEF240EL 19 9 DVPAPPPVD 18 68 DKQETDVHF 18 126 VLVLQVWDY 18 201 DVEREAQEA 18 323 LVFYTIPGQ 18 4 DIFPQDVPA 17 200 EDVEREAQE 17 286 WFVNPLKTF 17 138 SANDFLGSL 16 192 PVVKLKEAE 16 240 LTGKVEAEF 16 30 VVIWNTEDV 15 36 EDVVLDDEN 15 49 EMSSDIYVK 15 55 YVKSWV60K 15 254 LTVEEAEKR 15 304 RTLVLLLL3 15 306 LVLLLL300 15 326 YTIPGQISQ 15 334 QVIFRPLHK 15

TABLE XXVII 158P3D2v.1-B0702-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 249 240ELLTVE 1761625413 124 20VLVLQVW 1761625412 186 180GWWPVV 1381322590 123 120VLVLQV 1381322588 188 0GWWPVVKL 1346719875 313 00VLLTVFL 1346719873 314 0VLLTVFLL 1346719870 251 0ELLTVEEA 1346719866 125 0VLVLQVWD 1346719861 62 0KGLEHDKQ 1346719860 312 300VLLTVF 1315204205 187 80GWWPVVK 609485073 250 40ELLTVEE 2911588 10 VPAPPPVDI 23 276 KPSRPKTSF 23 159 GPELCSVQL 22 289 NPLKTFVFF 21 120 QPA120VLV 19 328 IPGQISQVI 19 22 QPISYELRV 18 96 LPTEREVSV 18 279 RPKTSFNWF 18 170 NGAGPRCNL 17 19 KPRQPISYE 16 191 WPVVKLKEA 16 266 KGRKQPEPL 15 300 RRYWRTLVL 15 302 YWRTLVLLL 15 32 IWNTEDVVL 14 119 RQPA120VL 14 121 PA120VLVL 14 154 VRGARGPEL 14 232 DMGGNVYIL 14 262 RPVGKGRKQ 14 12 APPPVDIKP 13 59 WV60KGLEH 13 71 ETDVHFNSL 13 105 WRRSGPFAL 13 142 FLGSLELQL 13 150 LPDMVRGAR 13 220 RRKGRPEDL 13 298 IWRRYWRTL 13 301 RYWRTLVLL 13 309 LLL300VLL 13 315 VLLTVFLLL 13 332 ISQVIFRPL 13 6 FPQDVPAPP 12 16 VDIKPRQPI 12 57 KSWV60KGL 12 88 RFVFRFDYL 12 109 GPFALEEAE 12 140 NDFLGSLEL 12 173 GPRCNLFRC 12 222 KGRPEDLEF 12 246 AEF240ELL 12 270 QPEPLEKPS 12 283 SFNWFVNPL 12 303 WRTLVLLLL 12

TABLE XXVIII 158P3D2v.1-B08-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 187 80GWWPVVK 1755316491 188 0GWWPVVKL 1441804 313 00VLLTVFL 1441804 314 0VLLTVFLL 1441804 251 0ELLTVEEA 1441795 125 0VLVLQVWD 1441794 62 0KGLEHDKQ 1441793 250 40ELLTVEE 262147 312 300VLLTVF 65809 220 RRKGRPEDL 28 181 CRRLR180G 25 193 VVKLKEAED 23 290 PLKTFVFFI 23 17 DIKPRQPIS 22 183 RLR180GWW 22 279 RPKTSFNWF 22 211 AGKKKRKQR 21 154 VRGARGPEL 20 213 KKKRKQRRR 20 272 EPLEKPSRP 20 159 GPELCSVQL 19 171 GAGPRCNLF 19 191 WPVVKLKEA 19 212 GKKKRKQRR 19 239 ILTGKVEAE 19 264 VGKGRKQPE 19 88 RFVFRFDYL 18 120 QPA120VLV 18 176 CNLFRCRRL 18 195 KLKEAEDVE 18 215 KRKQRRRKG 18 245 EAEF240EL 18 288 VNPLKTFVF 18 300 RRYWRTLVL 18 59 WV60KGLEH 17 96 LPTEREVSV 17 105 WRRSGPFAL 17 142 FLGSLELQL 17 298 IWRRYWRTL 17 302 YWRTLVLLL 17 308 LLLL300VL 17 309 LLL300VLL  17 315 VLLTVFLLL 17 27 ELRVVIWNT 16 103 SVWRRSGPF 16 138 SANDFLGSL 16 218 QRRRKGRPE 16 262 RPVGKGRKQ 16 266 KGRKQPEPL 16 277 PSRPKTSFN 16 53 DIYVKSWV6 15 115 EAEFRQPA1 15 121 PA120VLVL 15 258 EAEKRPVGK 15 10 VPAPPPVDI 14 178 LFRCRRLR1 14 210 QAGKKKRKQ 14 217 KQRRRKGRP 14 257 EEAEKRPVG 14 320 FLLLVFYTI 14

TABLE XXIX 158P3D2v.1-B1510-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 249 240ELLTVE 1761625414 124 20VLVLQVW 1761625413 186 180GWWPVV 1381322583 123 120VLVLQV 1381322581 188 0GWWPVVKL 1346719873 313 00VLLTVEL 1346719871 314 00VLLTVFL 1346719870 125 0VLVLQVWD 1346719862 251 0ELLTVEEA 1346719861 62 0KGLEHDKQ 1346719859 312 300VLLTVF 1315204206 187 80GWWPVVK 609485074 250 40ELLTVEE 2911589 32 IWNTEDVVL 16 298 IWRRYWRTL 15 332 ISQVIFRPL 15 159 GPELCSVQL 14 170 NGAGPRCNL 14 176 CNLFRCRRL 14 245 EAEF240EL 14 20 PRQPISYEL 13 57 KSWV60KGL 13 105 WRRSGPFAL 13 119 RQPA120VL 13 121 PA120VLVL 13 232 DMGGNVYIL 13 66 EHDKQETDV 12 71 ETDVHFNSL 12 140 NDFLGSLEL 12 154 VRGARGPEL 12 220 RRKGRPEDL 12 266 KGRKQPEPL 12 300 RRYWRTLVL 12 301 RYWRTLVLL 12 302 YWRTLVLLL 12 308 LLLL300VL 12 309 LLL300VLL 12 38 VVLDDENPL 11 138 SANDFLGSL 11 283 SFNWFVNPL 11 74 VHFNSLTGE 10 83 GNFNWRFVF 10 88 RFVFRFDYL 10 135 DRISANDFL 10 142 FLGSLELQL 10 246 AEF240ELL 10 303 WRTLVLLLL 10 315 VLLTVFLLL 10 42 DENPLTGEM 9 68 DKQETDVHF 9 81 GEGNFNWRF 9 85 FNWRFVFRF 9 171 GAGPRCNLF 9 222 KGRPEDLEF 9 276 KPSRPKTSF 9 289 NPLKTFVFF 9 24 ISYELRVVI 8 110 PFALEEAEF 8 145 SLELQLPDM 8 240 LTGKVEAEF 8 286 WFVNPLKTF 8 288 VNPLKTFVF 8 317 LTVFLLLVF 8 329 PGQISQVIF 8

TABLE XXX 158P3D2v.1-B2705-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 249 240ELLTVE 1761625417 124 20VLVLQVW 1761625416 186 180GWWPVV 1381322587 123 120VLVLQV 1381322583 188 0GWWPVVKL 1346719874 313 00VLLTVFL 1346719873 314 OVLLTVFLL 1346719873 125 OVLVLQVWD 1346719865 251 0ELLTVEEA 1346719864 62 0KGLEHDKQ 1346719863 312 300VLLTVF 1315204212 187 80GWWPVVK 609485081 250 40ELLTVEE 2911589 300 RRYWRTLVL 28 261 KRPVGKGRK 27 220 RRKGRPEDL 26 20 PRQPISYEL 25 99 EREVSVWRR 25 303 WRTLVLLLL 24 135 DRISANDFL 23 105 WRRSGPFAL 22 154 VRGARGPEL 22 87 WRFVFRFDY 21 174 PRCNLFRCR 21 219 RRRKGRPED 20 140 NDFLGSLEL 19 185 R180GWWPV 19 92 RFDYLPTER 18 106 RRSGPFALE 18 119 RQPA120VL 18 182 RRLR180GW 18 235 GNVYILTGK 18 268 RKQPEPLEK 18 81 GEGNFNWRF 17 83 GNFNWRFVF 17 159 GPELCSVQL 17 175 RCNLFRCRR 17 209 AQAGKKRK 17 213 KKKRKQRRR 17 222 KGRPEDLEF 17 267 GRKQPEPLE 17 276 KPSRPKTSF 17 293 TFVFFIWRR 17 330 GQISQVIFR 17 77 NSLTGEGNF 16 91 FRFDYLPTE 16 168 ARNGAGPRC 16 189 FWWPVVKLK 16 214 KKRKQRRRK 16 215 KRKQRRRKG 16 246 AEF240ELL 16 254 LTVEEAEKR 16 279 RPKTSFNWF 16 292 KTFVFFIWR 16 301 RYWRTLVLL 16 21 RQPISYELR 15 32 IWNTEDVVL 15 49 EMSSDIYVK 15 88 RFVFRFDYL 15 111 FALEEAEFR 15 121 PA120VLVL 15 147 ELQLPDMVR 15 157 ARGPELCSV 15 170 NGAGPRCNL 15 171 GAGPRCNLF 15 208 EAQAGKKKR 15 211 AGKKKRKQR 15 223 GRPEDLEFT 15 240 LTGKVEAEF 15 271 PEPLEKPSR 15 278 SRPKTSFNW 15 289 NPLKTFVFF 15 317 LTVFLLLVF 15 318 TVFLLLVFY 15 337 FRPLHK328 15 28 LRVVIWNTE 14 98 TEREVSVWR 14 110 PFALEEAEF 14 161 ELCSVQLAR 14 176 CNLFRCRRL 14 196 LKEAEDVER 14 205 EAQEAQAGK 14 206 AQEAQAGKK 14 207 QEAQAGKKK 14 216 RKQRRRKGR 14 232 DMGGNVYIL 14 266 KGRKQPEPL 14 273 PLEKPSRPK 14 286 WFVNPLKTF 14 294 FVFFIWRRY 14 308 LLLL300VL 14 315 VLLTVFLLL 14 329 PGQISQVIF 14

TABLE XXXI 158P3D2v.1-B2709-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 187 80GWWPVVK 1755316491 314 0VLLTVFLL 1441805 188 0GWWPVVKL 1441804 313 00VLLTVEL 1441804 125 0VLVLQVWD 1441795 251 0ELLTVEEA 1441795 62 0KGLEHDKQ 1441792 250 40ELLTVEE 262145 312 300VLLTVF 65810 300 RRYWRTLVL 27 185 R180GWWPV 24 220 RRKGRPEDL 24 20 PRQPISYEL 23 135 DRISANDFL 22 303 WRTLVLLLL 22 105 WRRSGPFAL 21 154 VRGARGPEL 21 157 ARGPELCSV 20 118 FRQPA120V 19 299 WRRYWRTLV 18 182 RRLR180GW 16 88 RFVFRFDYL 15 159 GPELCSVQL 15 219 RRRKGRPED 15 246 AEF240ELL 15 301 RYWRTLVLL 15 106 RRSGPFALE 14 119 RQPA120VL 14 121 PA120VLVL 14 223 GRPEDLEFT 14 267 GRKQPEPLE 14

TABLE XXX-II 158P3DWv.1-B4402-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 124 20VLVLQVW 1761625424 249 240ELLTVE 1761625414 123 120VLVLQV 1381322582 186 180GWWPVV 1381322581 314 0VLLTVFLL 1346719873 188 0GWWPVVKL 1346719872 251 0ELLTVEEA 1346719872 313 00VLLTVFL 1346719872 62 0KGLEHDKQ 1346719861 125 0VLVLQVWD 1346719861 312 300VLLTVF 1315204209 187 80GWWPVVK 609485072 250 40ELLTVEE 2911588 246 AEF240ELL 28 81 GEGNFNWRF 21 116 AEFRQPA12 20 199 AEDVEREAQ 17 57 KSWV60KGL 16 171  GAGPRCNLF 16 259 AEKRPVGKG 16 25 SYELRVVIW 15 51 SSDIYVKSW 15 71 ETDVHFNSL 15 83 GNFNWRFVF 15 140 NDFLGSLEL 15 222 KGRPEDLEF 15 274 LEKPSRPKT 15 286 WFVNPLKTF 15 315 VLLTVFLLL 15 318 TVFLLLVFY 15 18 IKPRQPISY 14 20 PRQPISYEL 14 26 YELRVVIWN 14 42 DENPLTGEM 14 121 PA120VLVL 14 245 EAEF240EL 14 257 EEAEKRPVG 14 288 VNPLKTFVF 14 289 NPLKTFVFF 14 294 FVFFIWRRY 14 295 VFFIWRRYW 14 300 RRYWRTLVL 14 301 RYWRTLVLL 14 302 YWRTLVLLL 14 303 WRTLVLLLL 14 309 LLL300VLL 14

TABLE XXXIII 158P3D2v.1-B5101-9-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 187 80GWWPVVK 1755316495 188 0GWWPVVKL 1441810 314 0VLLTVFLL 1441803 313 00VLLTVFL 1441799 251 0ELLTVEEA 1441797 125 0VLVLQVWD 1441795 62 0KGLEHDKQ 1441794 250 40ELLTVEE 262145 312 300VLLTVF 65807 328 IPGQISQVI 25 121 PA120VLVL 24 10 VPAPPPVDI 23 96 LPTEREVSV 23 24 ISYELRVVI 22 22 QPISYELRV 21 120 QPA120VLV 19 138 SANDFLGSL 18 320 FLLLVFYTI 18 159 GPELCSVQL 17 272 EPLEKPSRP 17 289 NPLKTFVFF 17 311 L300VLLTV 17 324 VFYTIPGQI 17 12 APPPVDIKP 16 94 DYLPTEREV 16 111 FALEEAEFR 16 245 EAEF240EL 16 6 FPQDVPAPP 15 46 LTGEMSSDI 15 82 EGNFNWRFV 15 210 QAGKKKRKQ 15 236 NVYILTGKV 15 248 F240ELLTV 15 262 RPVGKGRKQ 15 31 VIWNTEDVV 14 44 NPLTGEMSS 14 150 LPDMVRGAR 14 170 NGAGPRCNL 14 198 EAEDVEREA 14 208 EAQAGKKKR 14 231 TDMGGNVYI 14 266 KGRKQPEPL 14 279 RPKTSFNWF 14 300 RRYWRTLVL 14 315 VLLTVFLLL 14 11 PAPPPVDIK 13 13 PPPVDIKPR 13 23 PISYELRVV 13 32 IWNTEDVVL 13 129 LQVWDYDRI 13 146 LELQLPDMV 13 167 LARNGAGPR 13 191 WPVVKLKEA 13 194 VKLKEAEDV 13 224 RPEDLEFTD 13 232 DMGGNVYIL 13 258 EAEKRPVGK 13 290 PLKTFVFFI 13 308 LLLL300VL 13

TABLE XXXIV 158P3D2v.1-A1-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 124 20VLVLQVWD 1761625411 249 240ELLTVEE 1761625411 186 180GWWPVVK 1381322581 123 120VLVLQVW 1381322580 125 0VLVLQVWDY 1346719875 314 0VLLTVFLLL 1346719870 188 0GWWPVVKLK 1346719862 313 00VLLTVFLL 1346719862 62 0KGLEHDKQE 1346719859 251 0ELLTVEEAE 1346719859 312 300VLLTVFL 1315204197 187 80GWWPVVKL 609485072 250 40ELLTVEEA 2911596 17 DIKPRQPISY 23 46 LTGEMSSDIY 21 317 LTVFLLLVFY 21 71 ETDVHFNSLT 19 39 VLDDENPLTG 18 230 FTDMGGNVYI 18 25 SYELRVVIWN 17 139 ANDFLGSLEL 17 229 EFTDMGGNVY 17 34 NTEDVVLDDE 16 51 SSDIYVKSWV 16 86 NWRFVFRFDY 16 97 PTEREVSVWR 16 112 ALEEAEFRQP 15 293 TFVFFIWRRY 15 47 TGEMSSDIYV 14 145 SLELQLPDMV 14 206 AQEAQAGKKK 14 270 QPEPLEKPSR 14 273 PLEKPSRPKT 14 159 GPELCSVQLA 13 224 RPEDLEFTDM 13 256 VEEAEKRPVG 13 258 EAEKRPVGKG 13 80 TGEGNFNWRF 12 115 EAEFRQPA12 12 189 GWWPVVKLKE 12 245 EAEF240ELL 12 304 RTLVLLLL30 12 326 YTIPGQISQV 12

TABLE XXXV 158P3D2v.1-A0201-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 187 80GWWPVVKL 1755316508 314 0VLLTVFLLL 1441809 313 00VLLTVFLL 1441806 125 0VLVLQVWDY 1441802 188 0GWWPVVKLK 1441796 251 0ELLTVEEAE 1441795 62 0KGLEHDKQE 1441791 250 40ELLTVEEA 262153 312 300VLLTVFL 65814 310 LL300VLLTV 37 122 A120VLVLQV 28 315 VLLTVFLLLV 28 95 YLPTEREVSV 26 308 LLLL300VLL 26 247 EF240ELLTV 25 309 LLL300VLLT 24 326 YTIPGQISQV 24 156 GARGPELCSV 23 307 VLLLL300VL 23 31 VIWNTEDVVL 22 297 FIWRRYWRTL 22 145 SLELQLPDMV 21 137 ISANDFLGSL 20 128 VLQVWDYDRI 19 153 MVRGARGPEL 19 231 TDMGGNVYIL 18 306 LVLLLL300V 18 327 TIPGQISQVI 18 331 QISQVIFRPL 18

TABLE XXXVI 158P3D2v.1-A0203-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 124 20VLVLQVWD 1761625411 249 240ELLTVEE 1761625411 123 120VLVLQVW 1381322579 186 180GWWPVVK 1381322579 251 0ELLTVEEAE 1346719868 62 0KGLEHDKQE 1346719859 125 0VLVLQVWDY 1346719859 188 0GWWPVVKLK 1346719859 313 00VLLTVFLL 1346719859 314 0VLLTVFLLL 1346719859 312 300VLLTVFL  1315204197 187 80GWWPVVKL 609485069 250 40ELLTVEEA 2911595 202 VEREAQEAQA 18 3 IDIFPQDVPA 10 103 SVWRRSGPFA 10 107 RSGPFALEEA 10 113 LEEAEFRQPA 10 130 QVWDYDRISA 10 148 LQLPDMVRGA 10 159 GPELCSVQLA 10 163 CSVQLARNGA 10 190 WWPVVKLKEA 10 197 KEAEDVEREA 10 200 EDVEREAQEA 10 237 VYILTGKVEA 10 4 DIFPQDVPAP 9 104 VWRRSGPFAL 9 108 SGPFALEEAE 9 114 EEAEFRQPA1 9 131 VWDYDRISAN 9 149 QLPDMVRGAR 9 160 PELCSVQLAR 9 164 SVQLARNGAG 9 191 WPVVKLKEAE 9 198 EAEDVEREAQ 9 201 DVEREAQEAQ 9 203 EREAQEAQAG 9 238 YILTGKVEAE 9

TABLE XXXVII 158P3D2v.1-A3-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 124 20VLVLQVWD 1761625417 249 240ELLTVEE 1761625416 186 180GWWPVVK 1381322604 123 120VLVLQVW 1381322583 125 OVLVLQVWDY 1346719877 314 0VLLTVFLLL 1346719875 188 0GWWPVVKLK 1346719869 62 0KGLEHDKQE 1346719863 251 0ELLTVEEAE 1346719863 313 00VLLTVFLL 1346719860 312 300VLLTVFL 1315204201 187 80GWWPVVKL 609485073 250 40ELLTVEEA 2911586 183 RLR180GWWP 25 252 ELLTVEEAEK 25 166 QLARNGAGPR 24 195 KLKEAEDVER 23 122 A120VLVLQV 22 236 NVYILTGKVE 22 17 DIKPRQPISY 21 239 ILTGKVEAEF 21 287 FVNPLKTFVF 21 316 LLTVFLLLVF 21 149 QLPDMVRGAR 20 39 VLDDENPLTG 19 153 MVRGARGPEL 18 204 REAQEAQAGK 18 206 AQEAQAGKKK 18 213 KKKRKQRRRK 18 9 DVPAPPPVDI 17 23 PISYELRVVI 17 27 ELRVVIWNTE 17 30 VVIWNTEDVV 17 31 VIWNTEDVVL 17 103 SVWRRSGPFA 17 112 ALEEAEFRQP 17 136 RISANDFLGS 17 177 NLFRCRRLR1 17 247 EF240ELLTV 17 253 LLTVEEAEKR 17 257 EEAEKRPVGK 17 260 EKRPVGKGRK 17 307 VLLLL300VL 17 308 LLLL300VLL  17 310 LL300VLLTV 17 53 DIYVKSWV60 16 89 FVFRFDYLPT 16 95 YLPTEREVSV 16 127 LVLQVWDYDR 16 130 QVWDYDRISA 16 333 SQVIFRPLHK 16 334 QVIFRPLHK3 16 29 RVVIWNTEDV 15 45 PLTGEMSSDI 15 147 ELQLPDMVRG 15 193 VVKLKEAEDV 15 205 EAQEAQAGKK 15 243 KVEAEF240E 15 267 GRKQPEPLEK 15 297 FIWRRYWRTL 15 309 LLL300VLLT 15 315 VLLTVFLLLV 15 323 LVFYTIPGQI 15 10 VPAPPPVDIK 14 48 GEMSSDIYVK 14 64 GLEHDKQETD 14 101 EVSVWRRSGP 14 145 SLELQLPDMV 14 164 SVQLARNGAG 14 201 DVEREAQEAQ 14 221 RKGRPEDLEF 14 229 EFTDMGGNVY 14 259 AEKRPVGKGR 14 320 FLLLVFYTIP 14 327 TIPGQISQVI 14 2 WIDIFPQDVP 13 38 VVLDDENPLT 13 54 IYVKSWV60K 13 78 SLTGEGNFNW 13 157 ARGPELCSVQ 13 161 ELCSVQLARN 13 192 PVVKLKEAED 13 272 EPLEKPSRPK 13 306 LVLLLL300V 13

TABLE XXXVIII 158P3D2v.1-A26-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 187 80GWWPVVKL 1755316503 125 0VLVLQVWDY 1441816 314 0VLLTVFLLL 1441811 313 00VLLTVFLL 1441808 188 0GWWPVVKLK 1441796 62 0KGLEHDKQE 1441793 251 0ELLTVEEAE 1441793 250 40ELLTVEEA 262148 312 300VLLTVFL 65810 37 DVVLDDENPL 30 317 LTVFLLLVFY 26 17 DIKPRQPISY 25 229 EFTDMGGNVY 23 4 DIFPQDVPAP 22 73 DVHFNSLTGE 22 101 EVSVWRRSGP 21 46 LTGEMSSDIY 20 275 EKPSRPKTSF 20 334 QVIFRPLHK3 20 71 ETDVHFNSLT 19 153 MVRGARGPEL 19 201 DVEREAQEAQ 19 287 FVNPLKTFVF 19 318 TVFLLLVFYT 19 326 YTIPGQISQV 19 9 DVPAPPPVDI 18 82 EGNFNWRFVF 18 141 DFLGSLELQL 18 245 EAEF240ELL 18 282 TSFNWFVNPL 17 285 NWFVNPLKTF 17 133 DYDRISANDF 16 200 EDVEREAQEA 16 247 EF240ELLTV 16 258 EAEKRPVGKG 16 292 KTFVFFIWRR 16 323 LVFYTIPGQI 16 89 FVFRFDYLPT 15 161 ELCSVQLARN 15 293 TFVFFIWRRY 15

TABLE XXXIX 158P3D2v.1-B0702-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 249 240ELLTVEE 1761625414 124 20VLVLQVWD 1761625413 186 180GWWPVVK 1381322585 123 120VLVLQVW 1381322580 314 OVLLTVFLLL 1346719872 313 00VLLTVFLL 1346719870 62 0KGLEHDKQE 1346719860 125 0VLVLQVWDY 1346719860 188 0GWWPVVKLK 1346719860 251 0ELLTVEEAE 1346719860 312 300VLLTVFL 1315204211 187 80GWWPVVKL 609485083 250 40ELLTVEEA 2911592 120 QPA120VLVL 25 19 KPRQPISYEL 23 328 IPGQISQVIF 20 224 RPEDLEFTDM 19 289 NPLKTFVFFI 19 109 GPFALEEAEF 18 279 RPKTSFNWFV 18 22 QPISYELRVV 17 159 GPELCSVQLA 17 299 WRRYWRTLVL 16 139 ANDFLGSLEL 15 153 MVRGARGPEL 15 276 KPSRPKTSFN 15 12 APPPVDIKPR 14 141 DFLGSLELQL 14 169 RNGAGPRCNL 14 219 RRRKGRPEDL 14 231 TDMGGNVYIL 14 262 RPVGKGRKQP 14 301 RYWRTLVLLL 14 302 YWRTLVLLLL 14 331 QISQVIFRPL 14 10 VPAPPPVDIK 13 31 VIWNTEDVVL 13 104 VWRRSGPFAL 13 134 YDRISANDFL 13 158 RGPELCSVQL 13 173 GPRCNLFRCR 13 300 RRYWRTLVLL 13

TABLE XL 158P3D2v.1-B08-10-mers No Results Found.

TABLE XLI 158P3D2v.1-B1510-10-mers No Results Found.

TABLE XLII 158P3D2v.1- B2705-10-mers No Results Found.

TABLE XLIII 158P3D2v.1- B2709-10-mers No Results Found.

TABLE XLIV 158P3D2v.1-B4402-10-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 187 80GWWPVVKL 1755316503 314 0VLLTVFLLL 1441807 251 0ELLTVEEAE 1441806 313 00VLLTVFLL 1441805 125 0VLVLQVWDY 1441804 62 0KGLEHDKQE 1441796 188 0GWWPVVKLK 1441796 250 40ELLTVEEA 262146 312 300VLLTVFL 65814 70 QETDVHFNSL 23 244 VEAEF240EL 20 246 AEF240ELLT 19 116 AEFRQPA120 18 274 LEKPSRPKTS 17 285 NWFVNPLKTF 17 259 AEKRPVGKGR 16 17 DIKPRQPISY 15 26 YELRVVIWNT 15 48 GEMSSDIYVK 15 50 MSSDIYVKSW 15 114 EEAEFRQPA1 15 139 ANDFLGSLEL 15 199 AEDVEREAQE 15 229 EFTDMGGNVY 15 282 TSFNWFVNPL 15 287 FVNPLKTFVF 15 82 EGNFNWRFVF 14 104 VWRRSGPFAL 14 118 FRQPA120VL 14 120 QPA120VLVL 14 133 DYDRISANDF 14 141 DFLGSLELQL 14 160 PELCSVQLAR 14 170 NGAGPRCNLF 14 245 EAEF240ELL 14 257 EEAEKRPVGK 14 278 SRPKTSFNWF 14 288 VNPLKTFVFF 14 294 FVFFIWRRYW 14 301 RYWRTLVLLL 14 302 YWRTLVLLLL 14 308 LLLL300VLL 14 31 VIWNTEDVVL 13 42 DENPLTGEMS 13 56 VKSWV60KGL 13 87 WRFVFRFDYL 13 158 RGPELCSVQL 13 207 QEAQAGKKKR 13 225 PEDLEFTDMG 13 228 LEFTDMGGNV 13 231 TDMGGNVYIL 13 271 PEPLEKPSRP 13 275 EKPSRPKTSF 13 277 PSRPKTSFNW 13 297 FIWRRYWRTL 13 299 WRRYWRTLVL 13 300 RRYWRTLVLL 13 307 VLLLL300VL 13 316 LLTVFLLLVF 13 331 QISQVIFRPL 13 12 APPPVDIKPR 12 23 PISYELRVVI 12 24 ISYELRVVIW 12 37 DVVLDDENPL 12 76 FNSLTGEGNF 12 78 SLTGEGNFNW 12 81 GEGNFNWRFV 12 84 NFNWRFVFRF 12 86 NWRFVFRFDY 12 96 LPTEREVSVW 12 100 REVSVWRRSG 12 109 GPFALEEAEF 12 137 ISANDFLGSL 12 146 LELQLPDMVR 12 175 RCNLFRCRRL 12 182 RRLR180GWW 12 197 KEAEDVEREA 12 221 RKGRPEDLEF 12 256 VEEAEKRPVG 12 265 GKGRKQPEPL 12 290 PLKTFVFFIW 12 311 L300VLLTVF 12 317 LTVFLLLVFY 12 319 VFLLLVFYTI 12 323 LVFYTIPGQI 12

TABLE XLV 158P3D2v.1- B5101-10-mers No Results Found.

TABLE XLVII 58P3D2v.1-DRB10101-15-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the  length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 124 20VLVLQVWDYDRIS 1761625427 249 240ELLTVEEAEKRP 1761625412 123 120VLVLQVWDYDRI 1381322595 186 180GWWPVVKLKEAE 1381322580 188 0GWWPVVKLKEAEDV 1346719884 313 00VLLTVFLLLVFYT 1346719881 251 0ELLTVEEAEKRPVG 1346719878 314 0VLLTVFLLLVFYTI 1346719876 125 0VLVLQVWDYDRISA 1346719873 62 0KGLEHDKQETDVHF 1346719867 312 300VLLTVFLLLVFY 1315204214 187 80GWWPVVKLKEAED 609485086 250 40ELLTVEEAEKRPV 2911600 300 RRYWRTLVLLLL300 36 131 VWDYDRISANDFLGS 32 322 LLVFYTIPGQISQVI 32 285 NWFVNPLKTFVFFIW 31 305 TLVLLLL300VLLTV 31 73 DVHFNSLTGEGNFNW 29 143 LGSLELQLPDMVRGA 29 234 GGNVYILTGKVEAEF 28 245 EAEF240ELLTVEEA 28 325 FYTIPGQISQVIFRP 28 102 VSVWRRSGPFALEEA 27 181 CRRLR180GWWPVVK 27 191 WPVVKLKEAEDVERE 27 317 LTVFLLLVFYTIPGQ 27 27 ELRVVIWNTEDVVLD 26 151 PDMVRGARGPELCSV 26 227 DLEFTDMGGNVYILT 26 140 NDFLGSLELQLPDMV 25 237 VYILTGKVEAEF240 25 304 RTLVLLLL300VLLT 25 3 IDIFPQDVPAPPPVD 24 90 VFRFDYLPTEREVSV 24 101 EVSVWRRSGPFALEE 24 137 ISANDFLGSLELQLP 24 308 LLLL300VLLTVFLL 24 318 TVFLLLVFYTIPGQI 24 43 ENPLTGEMSSDIYVK 23 329 PGQISQVIFRPLHK3 23 2 WIDIFPQDVPAPPPV 22 4 DIFPQDVPAPPPVDI 22 7 PQDVPAPPPVDIKPR 22 12 APPPVDIKPRQPISY 22 120 QPA120VLVLQVWDY 22 156 GARGPELCSVQLARN 22 233 MGGNVYILTGKVEAE 22 310 LL300VLLTVFLLLV 22 321 LLLVFYTIPGQISQV 22 296 FFIWRRYWRTLVLLL 21 86 NWRFVFRFDYLPTER 20 108 SGPFALEEAEFRQPA 20 115 EAEFRQPA120VLVL 20 149 QLPDMVRGARGPELC 20 235 GNVYILTGKVEAEF2 20 284 FNWFVNPLKTFVFFI 20 13 PPPVDIKPRQPISYE 19 49 EMSSDIYVKSWV60K 19 52 SDIYVKSWV60KGLE 19 56 VKSWV60KGLEHDKQ 19 40 LDDENPLTGEMSSDI 25 82 EGNFNWRFVFRFDYL 19 84 NENWREVERFDYLPT 19 92 RFDYLPTEREVSVWR 19 161 ELCSVQLARNGAGPR 19 190 WWPVVKLKEAEDVER 19 283 SFNWFVNPLKTFVFF 19 35 TEDVVLDDENPLTGE 18 99 EREVSVWRRSGPFAL 18 110 PFALEEAEFRQPA12 18 117 EFRQPA120VLVLQV 18 134 YDRISANDFLGSLEL 18 139 ANDFLGSLELQLPDM 18 163 CSVQLARNGAGPRCN 18 198 EAEDVEREAQEAQAG 18 217 KQRRRKGRPEDLEFT 18 222 KGRPEDLEFTDMGGN 18 253 LLTVEEAEKRPVGKG 18 299 WRRYWRTLVLLLL30 18 303 WRTLVLLLL300VLL 18 306 LVLLLL300VLLTVF 18 311 L300VLLTVFLLLVF 18 331 QISQVIFRPLHK328 18

TABLE XLVII 158P3D2v.1-DRB10301-15-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the  length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 187 80GWWPVVKLKEAED 1755316499 62  0KGLEHDKQETDVHF 1441819 314 0VLLTVFLLLVFYTI 1441813 313 00VLLTVFLLLVFYT 1441806 251 0ELLTVEEAEKRPVG 1441805 125 0VLVLQVWDYDRISA 1441804 188 0GWWPVVKLKEAEDV 1441793 250 40ELLTVEEAEKRPV 262164 312 300VLLTVFLLLVFY 65825 35 TEDVVLDDENPLTGE 37 237 VYILTGKVEAEF240 34 36 EDVVLDDENPLTGEM 30 139 ANDFLGSLELQLPDM 26 306 LVLLLL300VLLTVF 24 47 TGEMSSDIYVKSWV6 23 305 TLVLLLL300VLLTV 21 308 LLLL300VLLTVFLL 21 15 PVDIKPRQPISYELR 20 29 RVVIWNTEDVVLDDE 20 245 EAEF240ELLTVEEA 20 285 NWFVNPLKTFVFFIW 20 3 IDIFPQDVPAPPPVD 19 76 FNSLTGEGNFNWRFV 19 88 RFVFRFDYLPTEREV 19 151 PDMVRGARGPELCSV 19 190 WWPVVKLKEAEDVER 19 227 DLEFTDMGGNVYILT 19 13 PPPVDIKPRQPISYE 18 21 RQPISYELRVVIWNT 18 135 DRISANDFLGSLELQ 18 162 LCSVQLARNGAGPRC 18 175 RCNLFRCRRLR180G 18 221 RKGRPEDLEFTDMGG 18 253 LLTVEEAEKRPVGKG 18 271 PEPLEKPSRPKTSFN 18 295 VFFIWRRYWRTLVLL 18 329 PGQISQVIFRPLHK3 18

TABLE XLVIII 158P3D2v.1-DRB10401-15-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 124 20VLVLQVWDYDRIS 1761625419 249 240ELLTVEEAEKRP 1761625411 123 120VLVLQVWDYDRI 1381322593 186 180GWWPVVKLKEAE 1381322585 188 0GWWPVVKLKEAEDV 1346719881 62 0KGLEHDKQETDVHF 1346719879 313 00VLLTVELLLVEYT 1346719879 125 0VLVLQVWDYDRISA 1346719873 251 0ELLTVEEAEKRPVG 1346719873 314 0VLLTVELLLVEYTI 1346719873 312 300VLLTVELLLVEY 1315204211 187 80GWWPVVKLKEAED 609485085 250 40ELLTVEEAEKRPV 2911605 281 KTSFNWFVNPLKTFV 28 300 RRYWRTLVLLLL300 28 21 RQPISYELRVVIWNT 26 36 EDVVLDDENPLTGEM 26 43 ENPLTGEMSSDIYVK 26 199 AEDVEREAQEAQAGK 26 111 FALEEAEFRQPA120 24 245 EAEF240ELLTVEEA 24 73 DVHFNSLTGEGNFNW 22 84 NFNWRFVFRFDYLPT 22 90 VFRFDYLPTEREVSV 22 92 RFDYLPTEREVSVWR 22 129 LQVWDYDRISANDFL 22 296 FFIWRRYWRTLVLLL 22 308 LLLL300VLLTVFLL 22 317 LTVFLLLVFYTIPGQ 22 322 LLVFYTIPGQISQVI 22 323 LVFYTIPGQISQVIF 22 27 ELRVVIWNTEDVVLD 20 35 TEDVVLDDENPLTGE 20 47 TGEMSSDIYVKSWV6 20 76 FNSLTGEGNFNWRFV 20 87 WRFVFRFDYLPTERE 20 93 FDYLPTEREVSVWRR 20 120 QPA120VLVLQVWDY 20 128 VLQVWDYDRISANDF 20 151 PDMVRGARGPELCSV 20 159 GPELCSVQLARNGAG 20 162 LCSVQLARNGAGPRC 20 241 TGKVEAEF240ELLT 20 253 LLTVEEAEKRPVGKG 20 271 PEPLEKPSRPKTSFN 20 285 NWFVNPLKTFVFFIW 20 292 KTFVFFIWRRYWRTL 20 304 RTLVLLLL300VLLT 20 305 TLVLLLL300VLLTV 20 316 LLTVFLLLVFYTIPG 20 319 VFLLLVFYTIPGQIS 20 325 FYTIPGQISQVIFRP 20 329 PGQISQVIFRPLHK3 20 59 WV60KGLEHDKQETD 18 67 HDKQETDVHFNSLTG 18 112 ALEEAEFRQPA120V 18 137 ISANDFLGSLELQLP 18 156 GARGPELCSVQLARN 18 161 ELCSVQLARNGAGPR 18 196 LKEAEDVEREAQEAQ 18 202 VEREAQEAQAGKKKR 18 233 MGGNVYILTGKVEAE 18 242 GKVEAEF240ELLTV 18 310 LL300VLLTVFLLLV 18 331 QISQVIFRPLHK328 18 3 IDIFPQDVPAPPPVD 16 30 VVIWNTEDVVLDDEN 16 52 SDIYVKSWV60KGLE 16 88 RFVFRFDYLPTEREV 16 102 VSVWRRSGPFALEEA 16 108 SGPFALEEAEFRQPA 16 115 EAEFRQPA120VLVL 16 131 VWDYDRISANDFLGS 16 139 ANDFLGSLELQLPDM 16 235 GNVYILTGKVEAEF2 16 284 FNWFVNPLKTFVFFI 16 291 LKTFVFFIWRRYWRT 16 293 TFVFFIWRRYWRTLV 16 299 WRRYWRTLVLLLL30 16 13 PPPVDIKPRQPISYE 15 7 PQDVPAPPPVDIKPR 14 25 SYELRVVIWNTEDVV 14 28 LRVVIWNTEDVVLDD 14 29 RVVIWNTEDVVLDDE 14 37 DVVLDDENPLTGEMS 14 99 EREVSVWRRSGPFAL 14 110 PFALEEAEFRQPA12 14 126 VLVLQVWDYDRISAN 14 134 YDRISANDFLGSLEL 14 140 NDFLGSLELQLPDMV 14 143 LGSLELQLPDMVRGA 14 147 ELQLPDMVRGARGPE 14 150 LPDMVRGARGPELCS 14 175 RCNLFRCRRLR180G 14 190 WWPVVKLKEAEDVER 14 193 VVKLKEAEDVEREAQ 14 225 PEDLEFTDMGGNVYI 14 230 FTDMGGNVYILTGKV 14 234 GGNVYILTGKVEAEF 14 288 VNPLKTFVFFIWRRY 14 303 WRTLVLLLL300VLL 14 306 LVLLLL300VLLTVF 14 307 VLLLL300VLLTVFL 14 318 TVFLLLVFYTIPGQI 14 320 FLLLVFYTIPGQISQ 14 321 LLLVFYTIPGQISQV 14

TABLE XLIX 158P3D2v.1-DRB11101-15-mers Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 187 80GWWPVVKLKEAED 1755316517 188 0GWWPVVKLKEAEDV 1441815 313 00VLLTVFLLLVFYT 1441805 125 0VLVLQVWDYDRISA 1441804 251 0ELLTVEEAEKRPVG 1441802 314 0VLLTVFLLLVFYTI 1441802 62 0KGLEHDKQETDVHF 1441798 250 40ELLTVEEAEKRPV 262156 312 300VLLTVFLLLVFY 65811 92 RFDYLPTEREVSVWR 25 84 NFNWRFVFRFDYLPT 24 235 GNVYILTGKVEAEF2 24 322 LLVFYTIPGQISQVI 23 147 ELQLPDMVRGARGPE 22 292 KTFVFFIWRRYWRTL 22 98 TEREVSVWRRSGPFA 21 99 EREVSVWRRSGP FAL 21 128 VLQVWDYDRISANDF 20 161 ELCSVQLARNGAGPR 20 178 LFRCRRLR180GWWP 20 140 NDFLGSLELQLPDMV 19 227 DLEFTDMGGNVYILT 19 293 TFVFFIWRRYWRTLV 19 296 FFIWRRYWRTLVLLL 19 300 RRYWRTLVLLLL300 19 305 TLVLLLL300VLLTV 19 318 TVFLLLVFYTIPGQI 19 3 IDIFPQDVPAPPPVD 18 90 VFRFDYLPTEREVSV 18 175 RCNLFRCRRLR180G 18 284 FNWFVNPLKTFVFFI 18 317 LTVFLLLVFYTIPGQ 18 86 NWRFVFRFDYLPTER 17 177 NLFRCRRLR180GWW 17 49 EMSSDIYVKSWV60K 16 67 HDKQETDVHFNSLTG 16 73 DVHFNSLTGEGNFNW 16 131 VWDYDRISANDFLGS 16 172 AGPRCNLFRCRRLR1 16 212 GKKKRKQRRRKGRPE 16 214 KKRKQRRRKGRPEDL 16 245 EAEF240ELLTVEEA 16 258 EAEKRPVGKGRKQPE 16 330 GQISQVIFRPLHK32 16 21 RQPISYELRVVIWNT 15 53 DIYVKSWV60KGLEH 15 59 WV60KGLEHDKQETD 15 253 LLTVEEAEKRPVGKG 15 254 LTVEEAEKRPVGKGR 15 271 PEPLEKPSRPKTSFN 15 13 PPPVDIKPRQPISYE 14 111 FALEEAEFRQPA120 14 145 SLELQLPDMVRGARG 14 148 LQLPDMVRGARGPEL 14 150 LPDMVRGARGPELCS 14 159 GPELCSVQLARNGAG 14 196 LKEAEDVEREAQEAQ 14 260 EKRPVGKGRKQPEPL 14 261 KRPVGKGRKQPEPLE 14 268 RKQPEPLEKPSRPKT 14 308 LLLL300VLLTVFLL 14 2 WIDIFPQDVPAPPPV 13 12 APPPVDIKPRQPISY 13 25 SYELRVVIWNTEDVV 13 34 NTEDVVLDDENPLTG 13 47 TGEMSSDIYVKSWV6 13 110 PFALEEAEFRQPA12 13 176 CNLFRCRRLR180GW 13 234 GGNVYILTGKVEAEF 13 281 KTSFNWFVNPLKTFV 13 285 NWFVNPLKTFVFFIW 13 291 LKTFVFFIWRRYWRT 13 316 LLTVFLLLVFYTIPG 13 319 VFLLLVFYTIPGQIS 13 329 PGQISQVIFRPLHK3 13

TABLE XXII 158P3D2 v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, A1-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 62 PQPGCPPAY 16 22 RHPGQPLVR 10 46 KPRPAPLSC 10 70 YTGTVLEQT 10 44 ASKPRPAPL 9 69 AYTGTVLEQ 9 21 RRHPGQPLV 8 30 RSVPSWSSS 8 36 SSSCGWAWA 8 52 LSCPRICCP 8 61 SPQPGCPPA 8 72 GTVLEQTLS 8 73 TVLEQTLSP 8

TABLE XXIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, A0201-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 24 PGQPLVRSV 16 21 RRHPGQPLV 15 44 ASKPRPAPL 15 66 CPPAYTGTV 15 27 PLVRSVPSW 14 70 YTGTVLEQT 14 20 LRRHPGQPL 13 73 TVLEQTLSP 13 12 SWQNCAFWL 12 71 TGTVLEQTL 12 61 SPQPGCPPA 11 19 WLRRHPGQP 10 36 SSSCGWAWA 10 49 PAPLSCPRI 10 56 RICCPSPQP 10 69 AYTGTVLEQ 10 42 AWASKPRPA 9 31 SVPSWSSSC 8 51 PLSCPRICC 8 52 LSCPRICCP 8 65 GCPPAYTGT 8 67 PPAYTGTVL 8 68 PAYTGTVLE 8

TABLE XXIV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, A0203-9-mers No Results Found.

TABLE XXV 158P3D2 v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, A3-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 19 WLRRHPGQP 21 28 LVRSVPSWS 19 22 RHPGQPLVR 18 73 TVLEQTLSP 18 31 SVPSWSSSC 16 38 SCGWAWASK 16 27 PLVRSVPSW 15 46 KPRPAPLSC 15 56 RICCPSPQP 15 44 ASKPRPAPL 13 30 RSVPSWSSS 12 48 RPAPLSCPR 12 14 QNCAFWLRR 11 51 PLSCPRICC 11

TABLE XXVI 158P3D2-v.18, ORF: 2932-4764, Frame +1, Part A, 610 aa, A26, 9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 73 TVLEQTLSP 15 31 SVPSWSSSC 14 70 YTGTVLEQT 13 28 LVRSVPSWS 12 71 TGTVLEQTL 12 72 GTVLEQTLS 12 62 PQPGCPPAY 11 10 RSSWQNCAF 9 12 SWQNCAFWL 9 20 LRRHPGQPL 9 27 PLVRSVPSW 9 44 ASKPRPAPL 9 67 PPAYTGTVL 9

TABLE XXVII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, B0702-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 67 PPAYTGTVL 24 63 QPGCPPAYT 21 61 SPQPGCPPA 19 66 CPPAYTGTV 18 20 LRRHPGQPL 17 23 HPGQPLVRS 16 46 KPRPAPLSC 16 44 ASKPRPAPL 15 48 RPAPLSCPR 14 26 QPLVRSVPS 13 54 CPRICCPSP 13 59 CPSPQPGCP 13 8 WPRSSWQNC 12 32 VPSWSSSCG 12 50 APLSCPRIC 12

TABLE XXVIII 158P3D2-v.18, ORF: 2932-4764, Frame +1 610 aa, Part A, B08-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 44 ASKPRPAPL 28 1 MCKRRWHWP 18 67 PPAYTGTVL 17 20 LRRHPGQPL 16 26 QPLVRSVPS 16

TABLE XXIX 158P3D2-v.18, ORF: 2932- 4764, Frame +1, 610 aa, Part A, B1510-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 22 RHPGQPLVR 14 44 ASKPRPAPL 13 67 PPAYTGTVL 13 71 TGTVLEQTL 13 6 WHWPRSSWQ 12 20 LRRHPGQPL 11 12 SWQNCAFWL 10 10 RSSWQNCAF 8

TABLE XXX 158P3D2 v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, B2705- 9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 20 LRRHPGQPL 21 21 RRHPGQPLV 20 22 RHPGQPLVR 18 48 RPAPLSCPR 17 3 KRRWHWPRS 15 4 RRWHWPRSS 15 10 RSSWQNCAF 15 29 VRSVPSWSS 14 40 GWAWASKPR 14 9 PRSSWQNCA 13 14 QNCAFWLRR 13 44 ASKPRPAPL 13 47 PRPAPLSCP 13 55 PRICCPSPQ 13 67 PPAYTGTVL 13 38 SCGWAWASK 12 71 TGTVLEQTL 12 2 CKRRWHWPR 11 12 SWQNCAFWL 11 13 WQNCAFWLR 11 15 NCAFWLRRH 11 49 PAPLSCPRI 11 62 PQPGCPPAY 11

TABLE XXXI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, B2709-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 21 RRHPGQPLV 24 20 LRRHPGQPL 20 3 KRRWHWPRS 14 4 RRWHWPRSS 14 10 RSSWQNCAF 12 44 ASKPRPAPL 12

TABLE XXXII 158P3D2-v.18, ORF: 2932-4764, Frame +1 610 aa, Part A, B4402 9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 44 ASKPRPAPL 18 62 PQPGCPPAY 15 10 RSSWQNCAF 13 12 SWQNCAFWL 13 35 WSSSCGWAW 13 11 SSWQNCAFW 12 27 PLVRSVPSW 12 5 RWHWPRSSW 11 67 PPAYTGTVL 11 71 TGTVLEQTL 11 20 LRRHPGQPL 10 33 PSWSSSCGW 10 49 PAPLSCPRI 10

TABLE XXIII 158P3D2-v.18, ORF: 2932- 4764, Frame +1, 610 aa, B5101-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 49 PAPLSCPRI 23 66 CPPAYTGTV 23 67 PPAYTGTVL 21 24 PGQPLVRSV 17 68 PAYTGTVLE 16 71 TGTVLEQTL 15 41 WAWASKPRP 14 50 APLSCPRIC 14 16 CAFWLRRHP 13 23 HPGQPLVRS 13 26 QPLVRSVPS 13 32 VPSWSSSCG 12 46 KPRPAPLSC 12

TABLE XXXIV 158P3D2-v.18, ORF: 2932-4764, Frame +1 610 aa, Part  A, A1-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 61 SPQPGCPPAY 21 72 GTVLEQTLSP 12 21 RRHPGQPLVR 11 44 ASKPRPAPLS 11

TABLE XXXV 158P3D2-v.18, ORF: 2932-4764, Frame +1 610 aa, Part A, A0201 10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 73 TVLEQTLSPL 22 19 WLRRHPGQPL 20 70 YTGTVLEQTL 18 23 HPGQPLVRSV 16 43 WASKPRPAPL 16 65 GCPPAYTGTV 15 11 SSWQNCAFWL 13 20 LRRHPGQPLV 13 48 RPAPLSCPRI 13 51 PLSCPRICCP 11 69 AYTGTVLEQT 11

TABLE XXXVI 158P3D2-v.18, ORF: 2932-4764, Frame +1 610 aa, Part A, A0203 10-mers No Results Found.

TABLE XXVII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610aa,  Part A, A3-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 19 WLRRHPGQPL 19 21 RRHPGQPLVR 19 27 PLVRSVPSWS 18 37 SSCGWAWASK 18 73 TVLEQTLSPL 16 28 LVRSVPSWSS 15 31 SVPSWSSSCG 14 56 RICCPSPQPG 13 30 RSVPSWSSSC 12 46 KPRPAPLSCP 12 61 SPQPGCPPAY 12 24 PGQPLVRSVP 11 4 RRWHWPRSSW 10 18 FWLRRHPGQP 10 22 RHPGQPLVRS 10 44 ASKPRPAPLS 10 45 SKPRPAPLSC 10 51 PLSCPRICCP 10 66 CPPAYTGTVL 10

TABLE XXXVIII 158P3D2-v.18, ORF: 2932-4764, Frame +1 610 aa, Part A, A26-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 73 TVLEQTLSPL 26 70 YTGTVLEQTL 17 72 GTVLEQTLSP 14 31 SVPSWSSSCG 13 61 SPQPGCPPAY 13

TABLE XXXIX 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, B0702 10-mers Each peptide is a portion of SEQ ID NO:31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 66 CPPAYTGTVL 23 48 RPAPLSCPRI 20 8 WPRSSWQNCA 18 23 HPGQPLVRSV 18 43 WASKPRPAPL 15 46 KPRPAPLSCP 14 59 CPSPQPGCPP 14 67 PPAYTGTVLE 14 19 WLRRHPGQPL 13 50 APLSCPRICC 13 54 CPRICCPSPQ 13 61 SPQPGCPPAY 13 26 QPLVRSVPSW 12 32 VPSWSSSCGW 12

TABLE XL 158P3D2- v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, B08-10-mers No Results Found.

TABLE XLI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, B1510-10-mers No Results Found.

TABLE XLII 158P3D2- v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, B2705-10-mers No Results Found.

TABLE XLIII 158P3D2- v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, B2709-10-mers No Results Found.

TABLE XLIV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, B4402-10-mers Each peptide is a portion of SEQ ID NO:31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 61 SPQPGCPPAY 17 34 SWSSSCGWAW 14 9 PRSSWQNCAF 13 11 SSWQNCAFWL 12 26 QPLVRSVPSW 12 43 WASKPRPAPL 12 66 CPPAYTGTVL 12 70 YTGTVLEQTL 12 73 TVLEQTLSPL 12 4 RRWHWPRSSW 11 10 RSSWQNCAFW 11 19 WLRRHPGQPL 11 32 VPSWSSSCGW 11 48 RPAPLSCPRI 11 44 ASKPRPAPLS 8

TABLE XLV 158P3D2- v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, B5101-10-mers No Results Found.

TABLE XLVI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, DRB1 0101-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fouteen. 26 QPLVRSVPSWSSSCG 30 17 AFWLRRHPGQPLVRS 26 16 CAFWLRRHPGQPLVR 24 25 GQPLVRSVPSWSSSC 23 57 ICCPSPQPGCPPAYT 22 71 TGTVLEQTLSPLWDE 22 23 HPGQPLVRSVPSWSS 21 69 AYTGTVLEQTLSPLW 19 32 VPSWSSSCGWAWASK 18 38 SCGWAWASKPRPAPL 18 40 GWAWASKPRPAPLSC 18 1 MCKRRWHWPRSSWQN 17 36 SSSCGWAWASKPRPA 17 42 AWASKPRPAPLSCPR 17 64 PGCPPAYTGTVLEQT 17 3 KRRWHWPRSSWQNCA 16 9 PRSSWQNCAFWLRRH 16 22 RHPGQPLVRSVPSWS 16 34 SWSSSCGWAWASKPR 16 41 WAWASKPRPAPLSCP 16 46 KPRPAPLSCPRICCP 16 49 PAPLSCPRICCPSPQ 16 70 YTGTVLEQTLSPLWD 16 39 CGWAWASKPRPAPLS 15

TABLE XLVII 158P3D2- v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, DRB1 0301-15 -mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 17 AFWLRRHPGQPLVRS 21 71 TGTVLEQTLSPLWDE 21 7 HWPRSSWQNCAFWLR 16 68 PAYTGTVLEQTLSPL 16 26 QPLVRSVPSWSSSCG 13 29 VRSVPSWSSSCGWAW 12 54 CPRICCPSPQPGCPP 12 72 GTVLEQTLSPLWDEL 12 25 GQPLVRSVPSWSSSC 11

TABLE XLVIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, DRB1 0401-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 26 QPLVRSVPSWSSSCG 26 3 KRRWHWPRSSWQNCA 22 23 HPGQPLVRSVPSWSS 18 63 QPGCPPAYTGTVLEQ 18 69 AYTGTVLEQTLSPLW 18 15 NCAFWLRRHPGQPLV 17 38 SCGWAWASKPRPAPL 16 67 PPAYTGTVLEQTLSP 16 17 AFWLRRHPGQPLVRS 14 25 GQPLVRSVPSWSSSC 14 29 VRSVPSWSSSCGWAW 14 71 TGTVLEQTLSPLWDE 14 72 GTVLEQTLSPLWDEL 14

TABLE XLIX 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part A, DRB1 1101-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 38 SCGWAWASKPRPAPL 26 40 GWAWASKPRPAPLSC 24 22 RHPGQPLVRSVPSWS 23 14 QNCAFWLRRHPGQPL 21 15 NCAFWLRRHPGQPLV 18 26 QPLVRSVPSWSSSCG 18 16 CAFWLRRHPGQPLVR 16 69 AYTGTVLEQTLSPLW 16 13 WQNCAFWLRRHPGQP 15 25 GQPLVRSVPSWSSSC 14 67 PPAYTGTVLEQTLSP 14

TABLE XXII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B A1-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 GISRQLLKH 10

TABLE XXIII 158P3D2-v.18, ORF:2932-4764, Frame +1, 610 aa, Part B, A0201-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 GISRQLLKH 16 6 LLKHNFDED 13 5 QLLKHNFDE 12

TABLE XXIV 158P3D2- v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, A0203-9-mers No Results Found.

TABLE XXIV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa,  Part B, A3-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 GISRQLLKH 18 5 QLLKHNFDE 14 6 LLKHNFDED 11

TABLE XXVI 158P3D2-v.18, ORF: 2932- 4764, Frame +1, 610 aa, Part B, A26-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 GISRQLLKH 10 3 SRQLLKHNF 10 2 ISRQLLKHN 5

TABLE XXVII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B B0702-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 8 KHNFDEDEM 7 3 SRQLLKHNF 6 1 GISRQLLKH 4 6 LLKHNFDED 17 4 RQLLKHNFD 11 3 SRQLLKHNF 9

TABLE XXVIII 158P3D2-v.18, ORF: 2932- 4764, Frame +1, 610 aa, Part B, B08-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 6 LLKHNFDED 17 4 RQLLKHNFD 11 3 SRQLLKHNF 9

TABLE XXIX 158P3D2-v.18, ORF: 2932- 4764, Frame +1, 610 aa, Part B, B1510-9-mers Each peptide is a portion of SEQ ID NO:31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 8 KHNFDEDEM 17

TABLE XXX 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, B2705-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 SRQLLKHNF 25 1 GISRQLLKH 15

TABLE XXXI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, B2709-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 SRQLLKHNF 19 8 KHNFDEDEM 11

TABLE XXXII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, B4402-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 SRQLLKHNF 13

TABLE XXXIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, B5101 9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 ISRQLLKHN 5 1 GISRQLLKH 4 4 RQLLKHNFD 4 5 QLLKHNFDE 4 6 LLKHNFDED 3

TABLE XXXIV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, A1-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 1 AGISRQLLKH 9

TABLE XXXV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, A0201-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 6 QLLKHNFDED 14 2 GISRQLLKHN 12 1 AGISRQLLKH 11 7 LLKHNFDEDE 11 8 LKHNFDEDEM 8

TABLE XXXVI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, A0203-10-mers No Results Found.

TABLE XXXVII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, A3-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 6 QLLKHNFDED 14 1 AGISRQLLKH 13 2 GISRQLLKHN 11 7 LLKHNFDEDE 11 3 ISRQLLKHNF 9 5 RQLLKHNFDE 7

TABLE XXXVIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, A26-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 1 AGISRQLLKH 11 2 GISRQLLKHN 8 3 ISRQLLKHNF 8

TABLE XXXIX 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, B0702-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 3 ISRQLLKHNF 9 8 LKHNFDEDEM 6

TABLE XL 158P3D2- v.18, ORF: 2932- 4764, Frame +1, 610 aa, Part B, B08-10-mers No Results Found.

TABLE XLI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, B1510-10-mers No Results Found.

TABLE XLVI 158P3D2- v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, DRB1 0101-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen.

TABLE XLII 158P3D2- v.18, ORF: 2932- 4764, Frame +1, 610 aa, Part B, B2705- 10-mers No Results Found.

TABLE XLIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, B2709- 10-mers No Results Found.

TABLE XLIV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, B4402-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 3 ISRQLLKHNF 11 1 AGISRQLLKH 9

TABLE XLV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, B5101- 10-mers No Results Found.

TABLE XLVI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, DRB1 0101-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 14 KHNFDEDEMDDPGDS 19 6 AGISRQLLKHNFDED 18 1 PFLAEAGISRQLLKH 15 5 EAGISRQLLKHNFDE 15 2 FLAEAGISRQLLKHN 14 9 SRQLLKHNFDEDEMD 10

TABLE XLVII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, DRB1 0301-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 14 KHNFDEDEMDDPGDS 20 2 FLAEAGISRQLLKHN 17 6 AGISRQLLKHNFDED 17 9 SRQLLKHNFDEDEMD 12 5 EAGISRQLLKHNFDE 11 10 RQLLKHNFDEDEMDD 11 12 LLKHNFDEDEMDDPG 11

TABLE XLVIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, DRB1 0401-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 2 FLAEAGISRQLLKHN 18 14 KHNFDEDEMDDPGDS 16 5 EAGISRQLLKHNFDE 14 1 PFLAEAGISRQLLKH 12 3 LAEAGISRQLLKHNF 12 7 GISRQLLKHNFDEDE 12 11 QLLKHNFDEDEMDDP 12

TABLE XLIX 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part B, DRB1 1101-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 14 KHNFDEDEMDDPGDS 16 2 FLAEAGISRQLLKHN 15 6 AGISRQLLKHNFDED 14 7 GISRQLLKHNFDEDE 14

TABLE XXII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, A1-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 5 QYEVWVQQG 11 3 ASQYEVWVQ 7

TABLE XXIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, A0201-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 LASQYEVWV 19 1 GLASQYEVW 13

TABLE XXIV 158P3D2-v.18 ORF: 2932-4764, Frame +1, 610 aa, Part C, A0203- 9-mers No Results Found.

TABLE XXV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, A3-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 GLASQYEVW 15 4 SQYEVWVQQ 14 9 WVQQGPQEP 13 7 EVWVQQGPQ 11

TABLE XXVI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, A26-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 7 EVWVQQGPQ 21 9 WVQQGPQEP 10

TABLE XXVII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B0702-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 10 VQQGPQEPF 12 2 LASQYEVWV 10

TABLE XXVIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B08-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 GLASQYEVW 7 10 VQQGPQEPF 7 2 LASQYEVWV 5 4 SQYEVWVQQ 5

TABLE XXIX 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B1510-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 10 VQQGPQEPF 8 1 GLASQYEVW 4 2 LASQYEVWV 4 9 WVQQGPQEP 4

TABLE XXX 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B2705-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 10 VQQGPQEPF 15

TABLE XXXI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B2709-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 LASQYEVWV 9 10 VQQGPQEPF 8

TABLE XXXII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B4402-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 GLASQYEVW 11 10 VQQGPQEPF 11 6 YEVWVQQGP 10

TABLE XXXIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B1501-9-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 LASQYEVWV 22

TABLE XXXIV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, A1-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 6 QYEVWVQQGP 10 4 ASQYEVWVQQ 5

TABLE XXXV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, A0201-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 2 GLASQYEVWV 25

TABLE XXXVI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, A0203-10-mers No Results Found.

TABLE XXXVII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, A3-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 2 GLASQYEVWV 16 8 EVWVQQGPQE 16 10 WVQQGPQEPF 14 4 ASQYEVWVQQ 10 5 SQYEVWVQQG 9 1 CGLASQYEVW 8

TABLE XXXVIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, A26-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 8 EVWVQQGPQE 22 10 WVQQGPQEPF 18

TABLE XXXIX 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B0702-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 2 GLASQYEVWV 9 10 WVQQGPQEPF 7

TABLE XL 158P3D2- v.18, ORF: 2932- 4764, Frame +1, 610 aa, Part C, B08- 10-mers No Results Found.

TABLE XLI 158P3D2- v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B1510- 10-mers No Results Found.

TABLE XLII 158P3D2- v.18, ORF: 2932- 4764, Frame +1, 610 aa, Part C, B2705- 10-mers No Results Found.

TABLE XLIII 158P3D2- v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B2709- 10-mers No Results Found.

TABLE XLIV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B4402-10-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 1 CGLASQYEVW 12 7 YEVWVQQGPQ 10 10 WVQQGPQEPF 10

TABLE XLV 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, B5101- 10-mers No Results Found.

TABLE XLVI 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, DRB1 0101-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 5 NCGLASQYEVWVQQG 22 9 ASQYEVWVQQGPQEP 20 2 HRANCGLASQYEVWV 14

TABLE XLVII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, DRB1 0301-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 5 NCGLASQYEVWVQQG 18 1 HHRANCGLASQYEVW 9

TABLE XLVIII 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, DRB1 0401-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 3 RANCGLASQYEVWVQ 18 10 SQYEVWVQQGPQEPF 18 9 ASQYEVWVQQGPQEP 16 5 NCGLASQYEVWVQQG 14 2 HRANCGLASQYEVWV 12 7 GLASQYEVWVQQGPQ 12

TABLE XLIX 158P3D2-v.18, ORF: 2932-4764, Frame +1, 610 aa, Part C, DRB1 1101-15-mers Each peptide is a portion of SEQ ID NO: 31; each start position is specified, the length of the peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 9 ASQYEVWVQQGPQEP 19 8 LASQYEVWVQQGPQE 12

TABLE L Protein Characteristics of 158P3D2 v.17 Bioinformatic 158P3D2 var.17 Program URL on World Wide Web Outcome Protein length 2036 aa Transmembrane TM Pred .ch.embnet.org/ 1 TM helix 2003-2020; N-terminus intracellular region (type II) HMMTop .enzim.hu/hmmtop/ 1 TM helix 2003-2022; N-terminus intracellular (type II) Sosui .genome.ad.jp/SOSui/ 1 TM helix 1999-2021 TMHMM .cbs.dtu.dk/services/TMHMM 1 TM helix 2000-2022; N-terminus extracellular (type I) Signal Peptide Signal P .cbs.dtu.dk/services/SignalP/ None pI pI/MW tool .expasy.ch/tools/ 5.64 Molecular weight pI/MW tool .expasy.ch/tools/ 227.6 kDa Localization PSORT II psort.nibb.ac.jp/ 26% cytoplasmic, 17% vesicles of secretory system, 13% nuclear, 13% mitochondrial, 8% plasma membrane Motifs Pfam .sanger.ac.uk/Pfam/ C2 domain; amino acid transporter; 7TM chemoreceptor; bradykinin; glycosyl hydrolase Prints .biochem.ucl.ac.uk/ C2 domain; endolaptase Blocks .blocks.fhcrc.org/ C2 domain

TABLE LI Exon composition of transcript 158P3D2 v.1 Exon Number Start End Exon 1 1 836 Exon 2 837 922 Exon 3 923 1021 Exon 4 1022 1263 Exon 5 1264 1547 Exon 6 1548 1648 Exon 7 1649 1961

TABLE LII(a) Nucleotide sequence of transcript variant 158P3D2 v.2 (SEQ ID NO: 335) atcaaggccc tgggctggag gaagacatcc cagatccaga ggagctcgac tgggggtcca 60 agtactatgc gtcgctgcag gagctccagg ggcagcacaa ctttgatgaa gatgaaatgg 120 atgatcctgg agattcagat ggggtcaacc tcatttctat ggttggggag atccaagacc 180 agggtgaggc tgaagtcaaa ggcactgtgt ccccaaaaaa agcagttgcc accctgaaga 240 tctacaacag gtccctggag gaagaattta accactttga agactggctg aatgtgtttc 300 ctctgtaccg agggcaaggg ggccaggatg gaggtggaga agaggaagga tctggacacc 360 ttgtgggcaa gttcaagggc tccttcctca tttaccctga atcagaggca gtgttgttct 420 ctgagcccca gatctctcgg gggatcccac agaaccggcc catcaagctc ctggtcagag 480 tgtatgttgt aaaggctacc aacctggctc ctgcagaccc caatggcaaa gcagaccctt 540 acgtggtggt gagcgctggc cgggagcggc aggacaccaa ggaacgctac atccccaagc 600 agctcaaccc catctttgga gagatcctgg agctaagcat ctctctccca gctgagacgg 660 agctgacggt cgccgtattt gaacatgacc tcgtgggttc tgacgacctc atcggggaga 720 cccacattga tctggaaaac cgattctata gccaccacag agcaaactgt gggctggcct 780 cccagtatga agtgtgggtc cagcagggcc cacaggagcc attctgagtt tctggccaaa 840 cacattcaag ctcacattcc cttttgtgtc tccagatcct atgatttcat ggaaggggac 900 cctcccaccc accgccactg ccaaccaaga catagctcag tggtcaagac ttgggcttgg 960 gagtcgggat cctgtaacga atgtcacttg accgctttct ttttttatga aacagtctcg 1020 ctctgtctcc caggttggag tgcagtggca cgatctcggc tgactgcaac ctccacctcc 1080 tgggttcaag cgattctcct gcctcagcct ccccagtagc tgggattaca ggcgtgggcc 1140 cccatgtcca gctaattttt atattttcgc tctgtctccc aggttggagt gcagtggcac 1200 gatctcggct gactgcaacc tccacctcct gggttcaagc gattctcctg cctcagcctc 1260 cccagtagct gggattacag gcgtgggccc ccatgtccag ctaattttta tatttttagt 1320 agagacaggg tttcaccatg ttgtccaggc tggtcttgaa cccctgacct caagtgatcc 1380 acccacctct gcctcccaaa gtgctgggat tacaggtgtg agccaccatg ccaggccctc 1440 ttaacctctt caagtctgtt ttctcatctg caaaacagag gtaataagat cagtatcttc 1500 ttaatggaag cacctgggct acattttttt cattcattgt tatcataaat gaggactaac 1560 ctgtctcccg ttgggagttt tgaacctaga cctcatgtct tcatgacgtc atcactgccc 1620 caggcccagc tgtgtcccta caccagcccc agctgacgca tcttcttttt ctgcctgtag 1680 agatggttac aatgcctggc gtgatgcatt ctggccttcg cagatcctgg cggggctgtg 1740 ccaacgctgt ggcctccctg cccctgaata ccgagccggt gctgtcaagg tgggcagcaa 1800 agtcttcctg acaccaccgg agaccctgcc cccagggatc tcttcacatg tggattgaca 1860 tctttcctca agatgtgcct gctccacccc cagttgacat caagcctcgg cagccaatca 1920 gctatgagct cagagttgtc atctggaaca cggaggatgt ggttctggat gacgagaatc 1980 cactcaccgg agagatgtcg agtgacatct atgtgaagag ctgggtgaag gggttggagc 2040 atgacaagca ggagacagac gttcacttca actccctgac tggggagggg aacttcaatt 2100 ggcgctttgt gttccgcttt gactacctgc ccacggagcg ggaggtgagc gtctggcgca 2160 ggtctggacc ctttgccctg gaggaggcgg agttccggca gcctgcagtg ctggtcctgc 2220 aggatccctg gagttgcagc taccagacat ggtgcgtggg gcccggggcc ccgagctctg 2280 ctctgtgcag ctggcccgca atggggccgg gccgaggtgc aatctgtttc gctgccgccg 2340 cctgaggggc tggtggccgg tagtgaagct gaaggaggca gaggacgtgg agcgggaggc 2400 gcaggaggct caggctggca agaagaagcg aaagcagagg aggaggaagg gccggccaga 2460 agacctggag ttcacagaca tgggtggcaa tgtgtacatc ctcacgggca aggtggaggc 2520 agagtttgag ctgctgactg tggaggaggc cgagaaacgg ccagtgggga aggggcggaa 2580 gcagccagag cctctggaga aacccagccg ccccaaaact tccttcaact ggtttgtgaa 2640 cccgctgaag acctttgtct tcttcatctg gcgccggtac tggcgcaccc tggtgctgct 2700 gctactggtg ctgctcaccg tcttcctcct cctggtcttc tacaccatcc ctggccagat 2760 cagccaggtc atcttccgtc ccctccacaa gtgactctcg ctgaccttgg acactcaccc 2820 agggtgccaa cccttcaatg cctgctcctg gaagtctttc ttacccatgt gagctacccc 2880 agagtctagt gcttcctctg aataaaccta tcacagcc 2918

TABLE LIII(a) Nucleotide sequence alignment of 121P1F1 v.1 (SEQ ID NO: 336) and 158P3D2 v.2 (SEQ ID NO: 337)

TABLE LIV(a) Peptide sequences of protein coded by 158P3D2 v.2 >158P3D2 v.2A (SEQ ID NO: 338) MDDPGDSDGV NLISMVGEIQ DQGEAEVKGT VSPKKAVATL KIYNRSLEEE FNHFEDWLNV 60 FPLYRGQGGQ DGGGEEEGSG HLVGKFKGSF LIYPESEAVL FSEPQISRGI PQNRPIKLLV 120 RVYVVKATNL APADPNGKAD PYVVVSAGRE RQDTKERYIP KQLNPIFGEI LELSISLPAE 180 TELTVAVFEH DLVGSDDLIG ETHIDLENRF YSHHRANCGL ASQYEVWVQQ GPQEPF 236 >158P3D2 v.2B (SEQ ID NO: 339) MVRGARGPEL CSVQLARNGA GPRCNLFRCR RLRGWWPVVK LKEAEDVERE AQEAQAGKKK 60 RKQRRRKGRP EDLEFTDMGG NVYILTGKVE AEFELLTVEE AEKRPVGKGR KQPEPLEKPS 120 RPKTSFNWFV NPLKTFVFFI WRRYWRTLVL LLLVLLTVFL LLVFYTIPGQ ISQVIFRPLH 180 K 181

TABLE LV(a) Amino acid sequence alignment of 121P1F1 v.1 (SEQ ID NO: 340) and 158P3D2 v.2 (SEQ ID NO: 341) Score = 372 bits (956), Expect = e-103Identities = 181/181 (100%), Positives = 181/181 (100%) V.1: 148 MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK 207 MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK V.14: 1 MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK 60 V.1: 208 RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS 267 RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS V.14: 61 RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS 120 V.1: 268 RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH 327 RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH V.14: 121 RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH 180 V.1: 328 K 328 K V.14: 181 K 181 Note: Protein variant 158P3D2 v.2A does not share common sequence with protein 158P3D2 v.1.

TABLE LII(b) Nucleotide sequence of transcript variant 158P3D2 v.14 (SEQ ID NO: 342) caggtgggcg ggctggtggg cagaagggca gacgggcaga ggaagtgcca gtgccactgg 60 gaccatggct ctgacggtaa gcgtgcaacg actaacaggg ctgaccggca cccacgaccg 120 acaagtgaag ctcacctttc gaggctttac ccagaaaaca agaaaaattc actgtggtcc 180 agaagcagat atcggtgagc tgttccgatg gccccactat ggggctccac tggctgggga 240 gtgtctgtct gtgcaggtgg tcaactgcag ccgtgtattc agccttaggc ctctagggac 300 cctggtgatc tccctgcagc agctacagaa tgctgggcat ttggtgctac gggaagccct 360 agtggatgag aatcttcaag tgtccccgat ccaggtggag cttgacctga agtaccagcc 420 cccagagggc gctactggag cctggtcaga ggaggacttt ggggcaccca tccaggacag 480 cttcgagtta atcatcccca atgtgggctt ccaggaactg gagcctgggg aggcccagct 540 ggagcggcgg gcagtggctc taggccgcag gctagctcga agtctaggcc agcaggacga 600 tgaagagaat gagctggagc ttgagctgga gcaggacctg gatgatgagc ctgacgtgga 660 actttctggt gttatgttca gccccctcaa gagccgcgcc agggccctgg cccatgggga 720 tcccttccag gtgtccagag ctcaagactt ccaggtggga gtcactgtgc tggaagccca 780 gaaactggtg ggagtcaaca ttaaccccta tgtggccgtg caagtggggg ggcagcgccg 840 tgtgaccgcc acacagcgtg ggaccagttg ccccttctac aatgagtact tcttgttcga 900 atttcatgac acgcggcttc gtctccaaga cttgctgctg gagatcacgg tgagtggggt 960 aggggtgacc agtgtccttc agagaagggg ggatgagaaa gctgcaggac taacaccacc 1020 ttcccccaag gctttccatt cgcagaccct cccctttatg gccacccgga taggcacctt 1080 caggatggac ctgggcatca tcttggacca gccagatggc cagttctacc aaagatgggt 1140 tccgctgcat gatccccgag acacccgcgc cgggaccaag ggtttcatta aggtcacctt 1200 gtccgtgagg gcgcgcgggg acctgccccc tccaatgcta cccccggccc cagggcactg 1260 ttcggacatc gagaagaacc tgctcctgcc gcgcggggtg cccgccgaga ggccatgggc 1320 gcggctccgc gtgcgcctgt accgcgccga ggggcttccc gcgctgcgcc tggggctgct 1380 gggcagcctg gtccgcgccc tgcacgacca gcgcgtcctg gtggagccct atgtgcgggt 1440 gtctttcctg gggcaggagg gcgagacgtc ggtgagcgcc gaggcggcgg cgcccgaatg 1500 gaacgagcag ctgagcttcg tggagctctt cccgccgctg acgcgcagcc tccgcctgca 1560 gctgcgggac gacgcgcccc tggtcgacgc ggcactcgct acgcacgtgc cggacctgag 1620 gcggatctcc catccgggcc gcgcggcggg gtttaaccct accttcggcc cggcctgggt 1680 gcccctctat ggctcgcccc ccggcgcggg gctccgggat agtcttcaag gtctcaacga 1740 aggcgttggc caaggcattt ggttccgcgg ccgccttctg ctggctgtgt ccatgcaggt 1800 gttggaaggg agagctgaac ctgagcctcc ccaggcccag caggggtcca cgttgtcccg 1860 gctcacccga aagaagaaaa agaaagccag aagggatcag accccaaagg cggttccgca 1920 gcacttggac gccagccccg gtgccgaggg gcctgagatc ccccgtgcca tggaggtgga 1980 ggtggaggag ctgctgccgc tgccagagaa tgtcctggcg ccctgtgaag atttcctgct 2040 tttcggtgtg ctcttcgagg ccaccatgat cgaccccacc gtggcctccc agcccatcag 2100 cttcgagatc tccattggtc gcgcaggccg tctggaggag caattgggcc gagggtccag 2160 ggctggggag ggaactgagg gtgcagccgt ggaggctcag cctctgctgg gagccaggcc 2220 agaggaggag aaagaggagg aagaactggg gacccatgct cagcggcctg agcccatgga 2280 cggcagtggg ccatacttct gcttgcccct ctgtcactgc aagccatgca tgcatgtgtg 2340 gagttgctgg gaggaccaca cctggcgcct gcagagcagc aactgcgtgc gcaaagtggc 2400 cgagaggctg gaccaggggc tgcaggaggt tgagagactg cagcgcaagc cggggcctgg 2460 cgcctgtgca cagctcaagc aggcactgga agtactggtg gctgggagca gacagttttg 2520 ccacggtgcc gagcgcagga cgatgacccg gcccaatgcc ctggatcgat gccgagggaa 2580 actcctggtg cacagcctga accttttggc taagcaagga ctgcgacttc tacgcggcct 2640 gagacggcgc aatgtgcaaa agaaggtggc actggccaag aagctcctgg caaaactgcg 2700 ctttctggct gaggagcccc agccacccct ccccgatgtg ctggtctgga tgctcagcgg 2760 gcagcgccgt gtggcctggg cccggatccc tgcccaggat gtgctgttct ctgtggttga 2820 ggaggaacgg ggccgagact gtgggaagat ccagagtcta atgctcacgg cacccggggc 2880 agcccctggt gaggtctgtg ccaagctgga gctcttcctg cggctgggcc tgggcaagca 2940 agccaaggcc tgcacctctg agctgccccc ggatttgctg cccgagccct cagccgggct 3000 gccctccagc ctacaccggg acgactttag ctacttccaa ctccgggctc acttgtacca 3060 ggcccggggt gtgttggctg cagatgacag tggcctctcg gacccctttg ctcgagtcct 3120 catctctacc cagtgtcaga ccacacgggt cctggagcag acgctgagcc ctctgtggga 3180 tgaactcctg gtatttgagc agttgatcgt ggatgggagg agggagcacc tgcaggagga 3240 gcctccatta gtgatcatca atgtatttga ccacaataag tttggccccc ccgtgttcct 3300 gggcagggca ctggccgccc caagggtaaa gctgatggag gacccatacc aacgcccaga 3360 gttgcagttc ttccccctga ggaagggacc ctgggcagcc ggagagctca ttgccgcctt 3420 tcaactcatt gaactagact acagtggccg acttgagccc tcagtgccca gtgaggtgga 3480 gccccaggat ctggcacccc tggttgagcc ccactctgga cgcctgtccc ttccacccaa 3540 cgtgtgccca gtgctcaggg agttccgtgt tgaggtgctg ttctggggtc ttaggggact 3600 tggtcgtgtg catctgctcg aggtggagca gccccaggtt gtactggagg tggctgggca 3660 aggtgtggag tctgaggtcc tggccagcta ccgtgagagc cccaatttca ctgagcttgt 3720 caggcatctg acagtggact tgccggagca gccttacttg cagcctccac tcagcatctt 3780 ggtgattgag cgccgggcct ttggccacac agtccttgtg ggttcccaca ttgtccccca 3840 catgctgcga ttcacatttc ggggtcatga ggatcctcct gaggaggaag gagagatgga 3900 ggagacaggg gatatgatgc ccaagggacc tcaaggacag aagtccctgg atcccttctt 3960 ggctgaagcg ggtatatcca gacagctcct gaagcctcct ctgaagaagc tcccactagg 4020 aggcctccta aatcaaggcc ctgggctgga ggaagacatc ccagatccag aggagctcga 4080 ctgggggtcc aagtactatg cgtcgctgca ggagctccag gggcagcaca actttgatga 4140 agatgaaatg gatgatcctg gagattcaga tggggtcaac ctcatttcta tggttgggga 4200 gatccaagac caggatctac aacaggtccc tgaaggaaga atttaaccac tttgaagact 4260 ggctgaatgt gtttcctctg taccgagggc aagggggcca ggatggaggt ggagaagagg 4320 aaggatctgg acaccttgtg ggcaagttca agggctcctt cctcatttac cctgaatcag 4380 aggcagtgtt gttctctgag ccccagatct cccgggggat cccacagaac cggcccatca 4440 agctcctggt cagagtgtat gttgtaaagg ctaccaacct ggctcctgca gaccccaatg 4500 gcaaagcaga cccttacgtg gtggtgagcg ctggccggga gcggcaggac accaaggaac 4560 gctacatccc caagcagctc aaccccatct ttggagagat cctggagcta agcatctctc 4620 tcccagctga gacggagctg acggtcgccg tatttgatca tgacctcgtg ggttctgacg 4680 acctcatcgg ggagacccac attgatctgg aaaaccgatt ctatagccac cacagagcaa 4740 actgtgggct ggcctcccag tatgaagtgt gggtccagca gggcccacag gagccattct 4800 gagtttctgg ccaaacacat tcaagctcac attccctttt gtgtctccag atcctatgat 4860 ttcatggaag gggaccctcc cacccaccgc cactgccaac caagacatag ctcagtggtc 4920 aagacttggg cttgggagtc gggatcctgt aacgaatgtc acttgaccgc tttctttttt 4980 tatgaaacag tctcgctctg tctcccaggt tggagtgcag tggcacgatc tcggctgact 5040 gcaacctcca cctcctgggt tcaagcgatt ctcctgcctc agcctcccca gtagctggga 5100 ttacaggcgt gggcccccat gtccagctaa tttttatatt ttcgctctgt ctcccaggtt 5160 ggagtgcagt ggcacgatct cggctgactg caacctccac ctcctgggtt caagcgattc 5220 tcctgcctca gcctccccag tagctgggat tacaggcgtg ggcccccatg tccagctaat 5280 ttttatattt ttagtagaga cagggtttca ccatgttgtc caggctggtc ttgaacccct 5340 gacctcaagt gatccaccca cctctgcctc ccaaagtgct gggattacag gtgtgagcca 5400 ccatgccagg ccctcttaac ctcttcaagt ctgttttctc atctgcaaaa cagaggtaat 5460 aagatcagta tcttcttaat ggaagcacct ggactacatt tttttcattc attgttatca 5520 taaatgagga ctaacctgtc tcccgttggg agttttgaac ctagacctca tgtcttcatg 5580 acgtcatcac tgccccaggc ccagctgtgt ccctacacca gccccagctg acgcatcttc 5640 tttttctgcc tgtagagatg gttacaatgc ctggcgtgat gcattctggc cttcgcagat 5700 cctggcgggg ctgtgccaac gctgtggcct ccctgcccct gaataccgag ccggtgctgt 5760 caaggtgggc agcaaagtct tcctgacacc accggagacc ctgcccccag ggatctcttc 5820 acatgtggat tgacatcttt cctcaagatg tgcctgctcc acccccagtt gacatcaagc 5880 ctcggcagcc aatcagctat gagctcagag ttgtcatctg gaacacggag gatgtggttc 5940 tggatgacga gaatccactc accggagaga tgtcgagtga catctatgtg aagaggtagg 6000 ctgctggccg ggcggggcaa cggcggtgca ctagggggat tgcaaatggg tgtgggccct 6060 cgggctgagt ccagagcccc gaccccaggc cctccgtggt gctgagagcg gggtgaggag 6120 tgggttctcc atgtagctcc agccctgacg ctcacccacc ccggccccag ctgggtgaag 6180 gggttggagc atgacaagca ggagacagac gttcacttca actccctgac tggggagggg 6240 aacttcaatt ggcgctttgt gttccgcttt gactacctgc ccacggagcg ggaggtgagc 6300 gtctggcgca ggtctggacc ctttgccctg gaggaggcgg agttccggca gcctgcagtg 6360 ctggtcctgc aggtctggga ctatgaccgc atctctgcca atgacttcct tggatccctg 6420 gagttgcagc taccagacat ggtgcgtggg gcccggggcc ccgagctctg ctctgtgcag 6480 ctggcccgca atggggccgg gccgaggtgc aatctgtttc gctgccgccg cctgaggggc 6540 tggtggccgg tagtgaagct gaaggaggca gaggacgtgg agcgggaggc gcaggaggct 6600 caggctggca agaagaagcg aaagcagagg aggaggaagg gccggccaga agacctggag 6660 ttcacagaca tgggtggcaa tgtgtacatc ctcacgggca aggtggaggc agagtttgag 6720 ctgctgactg tggaggaggc cgagaaacgg ccagtgggga aggggcggaa gcagccagag 6780 cctctggaga aacccagccg ccccaaaact tccttcaact ggtttgtgaa cccgctgaag 6840 acctttgtct tcttcatctg gcgccggtac tggcgcaccc tggtgctgct gctactggtg 6900 ctgctcaccg tcttcctcct cctggtcttc tacaccatcc ctggccagat cagccaggtc 6960 atcttccgtc ccctccacaa gtgactctcg ctgaccttgg acactcaccc agggtgccaa 7020 cccttcaatg cctgctcctg gaagtctttc ttacccatgt gagctacccc agagtctagt 7080 gcttcctctg aataaaccta tcacagcc 7108

TABLE LIII(b) Nucleotide sequence alignment of 121P1F1 v.1 (SEQ ID NO: 343) and 158P3D2 v.14 (SEQ ID NO: 344)

TABLE LIV(b) Peptide sequences of protein coded by 158P3D2 v.14 (SEQ ID NO: 345) MALTVSVQRL TGLTGTHDRQ VKLTFRGFTQ KTRKIHCGPE ADIGELFRWP HYGAPLAGEC 60 LSVQVVNCSR VFSLRPLGTL VISLQQLQNA GHLVLREALV DENLQVSPIQ VELDLKYQPP 120 EGATGAWSEE DFGAPIQDSF ELIIPNVGFQ ELEPGEAQLE RRAVALGRRL ARSLGQQDDE 180 ENELELELEQ DLDDEPDVEL SGVMFSPLKS RARALAHGDP FQVSRAQDFQ VGVTVLEAQK 240 LVGVNINPYV AVQVGGQRRV TATQRGTSCP FYNEYFLFEF HDTRLRLQDL LLEITVSGVG 300 VTSVLQRRGD EKAAGLTPPS PKAFHSQTLP FMATRIGTFR MDLGIILDQP DGQFYQRWVP 360 LHDPRDTRAG TKGFIKVTLS VRARGDLPPP MLPPAPGHCS DIEKNLLLPR GVPAERPWAR 420 LRVRLYRAEG LPALRLGLLG SLVRALHDQR VLVEPYVRVS FLGQEGETSV SAEAAAPEWN 480 EQLSFVELFP PLTRSLRLQL RDDAPLVDAA LATHVPDLRR ISHPGRAAGF NPTFGPAWVP 540 LYGSPPGAGL RDSLQGLNEG VGQGIWFRGR LLLAVSMQVL EGRAEPEPPQ AQQGSTLSRL 600 TRKKKKKARR DQTPKAVPQH LDASPGAEGP EIPRAMEVEV EELLPLPENV LAPCEDFLLF 660 GVLFEATMID PTVASQPISF EISIGRAGRL EEQLGRGSRA GEGTEGAAVE AQPLLGARPE 720 EEKEEEELGT HAQRPEPMDG SGPYFCLPLC HCKPCMHVWS CWEDHTWRLQ SSNCVRKVAE 780 RLDQGLQEVE RLQRKPGPGA CAQLKQALEV LVAGSRQFCH GAERRTMTRP NALDRCRGKL 840 LVHSLNLLAK QGLRLLRGLR RRNVQKKVAL AKKLLAKLRF LAEEPQPPLP DVLVWMLSGQ 900 RRVAWARIPA QDVLFSVVEE ERGRDCGKIQ SLMLTAPGAA PGEVCAKLEL FLRLGLGKQA 960 KACTSELPPD LLPEPSAGLP SSLHRDDFSY FQLRAHLYQA RGVLAADDSG LSDPFARVLI 1020 STQCQTTRVL EQTLSPLWDE LLVFEQLIVD GRREHLQEEP PLVIINVFDH NKFGPPVFLG 1080 RALAAPRVKL MEDPYQRPEL QFFPLRKGPW AAGELIAAFQ LIELDYSGRL EPSVPSEVEP 1140 QDLAPLVEPH SGRLSLPPNV CPVLREFRVE VLFWGLRGLG RVHLLEVEQP QVVLEVAGQG 1200 VESEVLASYR ESPNFTELVR HLTVDLPEQP YLQPPLSILV IERRAFGHTV LVGSHIVPHM 1260 LRFTFRGHED PPEEEGEMEE TGDMMPKGPQ GQKSLDPFLA EAGISRQLLK PPLKKLPLGG 1320 LLNQGPGLEE DIPDPEELDW GSKYYASLQE LQGQHNFDED EMDDPGDSDG VNLISMVGEI 1380 QDQDLQQVPE GRI 1393

TABLE LV(b) Amino acid sequence alignment of 121P1F1 v.1 and 158P3D2 v.14 No significant similarity

TABLE LIV(b) Nucleotide sequence of transcript variant 158P3D2 v.14 (SEQ ID NO: 346) caggtgggcg ggctggtggg cagaagggca gacgggcaga ggaagtgcca gtgccactgg 60 gaccatggct ctgacggtaa gcgtgcaacg actaacaggg ctgaccggca cccacgaccg 120 acaagtgaag ctcacctttc gaggctttac ccagaaaaca agaaaaattc actgtggtcc 180 agaagcagat atcggtgagc tgttccgatg gccccactat ggggctccac tggctgggga 240 gtgtctgtct gtgcaggtgg tcaactgcag ccgtgtattc agccttaggc ctctagggac 300 cctggtgatc tccctgcagc agctacagaa tgctgggcat ttggtgctac gggaagccct 360 agtggatgag aatcttcaag tgtccccgat ccaggtggag cttgacctga agtaccagcc 420 cccagagggc gctactggag cctggtcaga ggaggacttt ggggcaccca tccaggacag 480 cttcgagtta atcatcccca atgtgggctt ccaggaactg gagcctgggg aggcccagct 540 ggagcggcgg gcagtggctc taggccgcag gctagctcga agtctaggcc agcaggacga 600 tgaagagaat gagctggagc ttgagctgga gcaggacctg gatgatgagc ctgacgtgga 660 actttctggt gttatgttca gccccctcaa gagccgcgcc agggccctgg cccatgggga 720 tcccttccag gtgtccagag ctcaagactt ccaggtggga gtcactgtgc tggaagccca 780 gaaactggtg ggagtcaaca ttaaccccta tgtggccgtg caagtggggg ggcagcgccg 840 tgtgaccgcc acacagcgtg ggaccagttg ccccttctac aatgagtact tcttgttcga 900 atttcatgac acgcggcttc gtctccaaga cttgctgctg gagatcacgg tgagtggggt 960 aggggtgacc agtgtccttc agagaagggg ggatgagaaa gctgcaggac taacaccacc 1020 ttcccccaag gctttccatt cgcagaccct cccctttatg gccacccgga taggcacctt 1080 caggatggac ctgggcatca tcttggacca gccagatggc cagttctacc aaagatgggt 1140 tccgctgcat gatccccgag acacccgcgc cgggaccaag ggtttcatta aggtcacctt 1200 gtccgtgagg gcgcgcgggg acctgccccc tccaatgcta cccccggccc cagggcactg 1260 ttcggacatc gagaagaacc tgctcctgcc gcgcggggtg cccgccgaga ggccatgggc 1320 gcggctccgc gtgcgcctgt accgcgccga ggggcttccc gcgctgcgcc tggggctgct 1380 gggcagcctg gtccgcgccc tgcacgacca gcgcgtcctg gtggagccct atgtgcgggt 1440 gtctttcctg gggcaggagg gcgagacgtc ggtgagcgcc gaggcggcgg cgcccgaatg 1500 gaacgagcag ctgagcttcg tggagctctt cccgccgctg acgcgcagcc tccgcctgca 1560 gctgcgggac gacgcgcccc tggtcgacgc ggcactcgct acgcacgtgc cggacctgag 1620 gcggatctcc catccgggcc gcgcggcggg gtttaaccct accttcggcc cggcctgggt 1680 gcccctctat ggctcgcccc ccggcgcggg gctccgggat agtcttcaag gtctcaacga 1740 aggcgttggc caaggcattt ggttccgcgg ccgccttctg ctggctgtgt ccatgcaggt 1800 gttggaaggg agagctgaac ctgagcctcc ccaggcccag caggggtcca cgttgtcccg 1860 gctcacccga aagaagaaaa agaaagccag aagggatcag accccaaagg cggttccgca 1920 gcacttggac gccagccccg gtgccgaggg gcctgagatc ccccgtgcca tggaggtgga 1980 ggtggaggag ctgctgccgc tgccagagaa tgtcctggcg ccctgtgaag atttcctgct 2040 tttcggtgtg ctcttcgagg ccaccatgat cgaccccacc gtggcctccc agcccatcag 2100 cttcgagatc tccattggtc gcgcaggccg tctggaggag caattgggcc gagggtccag 2160 ggctggggag ggaactgagg gtgcagccgt ggaggctcag cctctgctgg gagccaggcc 2220 agaggaggag aaagaggagg aagaactggg gacccatgct cagcggcctg agcccatgga 2280 cggcagtggg ccatacttct gcttgcccct ctgtcactgc aagccatgca tgcatgtgtg 2340 gagttgctgg gaggaccaca cctggcgcct gcagagcagc aactgcgtgc gcaaagtggc 2400 cgagaggctg gaccaggggc tgcaggaggt tgagagactg cagcgcaagc cggggcctgg 2460 cgcctgtgca cagctcaagc aggcactgga agtactggtg gctgggagca gacagttttg 2520 ccacggtgcc gagcgcagga cgatgacccg gcccaatgcc ctggatcgat gccgagggaa 2580 actcctggtg cacagcctga accttttggc taagcaagga ctgcgacttc tacgcggcct 2640 gagacggcgc aatgtgcaaa agaaggtggc actggccaag aagctcctgg caaaactgcg 2700 ctttctggct gaggagcaca actttgatga agatgaaatg gatgatcctg gagattcaga 2760 tggggtcaac ctcatttcta tggttgggga gatccaagac cagggtgagg ctgaagtcaa 2820 aggcactgtg tccccaaaaa aagcagttgc caccctgaag atctacaaca ggtccctgaa 2880 ggaagaattt aaccactttg aagactggct gaatgtgttt cctctgtacc gagggcaagg 2940 gggccaggat ggaggtggag aagaggaagg atctggacac cttgtgggca agttcaaggg 3000 ctccttcctc atttaccctg aatcagaggc agtgttgttc tctgagcccc agatctcccg 3060 ggggatccca cagaaccggc ccatcaagct cctggtcaga gtgtatgttg taaagctaag 3120 aaacctttgc aaaatccaag gtcatgaaga cttttgcctg ttttctgctg ctaccaacct 3180 ggctcctgca gaccccaatg gcaaagcaga cccttacgtg gtggtgagcg ctggccggga 3240 gcggcaggac accaaggaac gctacatccc caagcagctc aaccccatct ttggagagat 3300 cctggagcta agcatctctc tcccagctga gacggagctg acggtcgccg tatttgatca 3360 tgacctcgtg ggttctgacg acctcatcgg ggagacccac attgatctgg aaaaccgatt 3420 ctatagccac cacagagcaa actgtgggct ggcctcccag tatgaagtgt gggtccagca 3480 gggcccacag gagccattct gagtttctgg ccaaacacat tcaagctcac attccctttt 3540 gtgtctccag atcctatgat ttcatggaag gggaccctcc cacccaccgc cactgccaac 3600 caagacatag ctcagtggtc aagacttggg cttgggagtc gggatcctgt aacgaatgtc 3660 acttgaccgc tttctttttt tatgaaacag tctcgctctg tctcccaggt tggagtgcag 3720 tggcacgatc tcggctgact gcaacctcca cctcctgggt tcaagcgatt ctcctgcctc 3780 agcctcccca gtagctggga ttacaggcgt gggcccccat gtccagctaa tttttatatt 3840 ttcgctctgt ctcccaggtt ggagtgcagt ggcacgatct cggctgactg caacctccac 3900 ctcctgggtt caagcgattc tcctgcctca gcctccccag tagctgggat tacaggcgtg 3960 ggcccccatg tccagctaat ttttatattt ttagtagaga cagggtttca ccatgttgtc 4020 caggctggtc ttgaacccct gacctcaagt gatccaccca cctctgcctc ccaaagtgct 4080 gggattacag gtgtgagcca ccatgccagg ccctcttaac ctcttcaagt ctgttttctc 4140 atctgcaaaa cagaggtaat aagatcagta tcttcttaat ggaagcacct ggactacatt 4200 tttttcattc attgttatca taaatgagga ctaacctgtc tcccgttggg agttttgaac 4260 ctagacctca tgtcttcatg acgtcatcac tgccccaggc ccagctgtgt ccctacacca 4320 gccccagctg acgcatcttc tttttctgcc tgtagagatg gttacaatgc ctggcgtgat 4380 gcattctggc cttcgcagat cctggcgggg ctgtgccaac gctgtggcct ccctgcccct 4440 gaataccgag ccggtgctgt caaggtgggc agcaaagtct tcctgacacc accggagacc 4500 ctgcccccag ggatctcttc acatgtggat tgacatcttt cctcaagatg tgcctgctcc 4560 acccccagtt gacatcaagc ctcggcagcc aatcagctat gagctcagag ttgtcatctg 4620 gaacacggag gatgtggttc tggatgacga gaatccactc accggagaga tgtcgagtga 4680 catctatgtg aagaggtagg ctgctggccg ggcggggcaa cggcggtgca ctagggggat 4740 tgcaaatggg tgtgggccct cgggctgagt ccagagcccc gaccccaggc cctccgtggt 4800 gctgagagcg gggtgaggag tgggttctcc atgtagctcc agccctgacg ctcacccacc 4860 ccggccccag ctgggtgaag gggttggagc atgacaagca ggagacagac gttcacttca 4920 actccctgac tggggagggg aacttcaatt ggcgctttgt gttccgcttt gactacctgc 4980 ccacggagcg ggaggtgagc gtctggcgca ggtctggacc ctttgccctg gaggaggcgg 5040 agttccggca gcctgcagtg ctggtcctgc aggtctggga ctatgaccgc atctctgcca 5100 atgacttcct tggtattaca atgcttagcc ttccccaccc tcagcccctg cctccagccc 5160 tcacctccgc ccctgcctcc agccctcact tccgtccccc agttccctac tctgacccaa 5220 ccttgaatct tgggattttg gacccgaggt gtgaaacctt tgctttctgg cctaattact 5280 gagttaatta ggcctagacc acagtaacct ccattcccac ccagagtctc tgattcaact 5340 ctgatttgac cctagcttgt caccctgaca ccgactccac agcctttggt ccttggcact 5400 ctgatcccga cccttggccc tcttccactg ggaagtagca atgggtggac cgctgggctg 5460 tggtctgggt ggtctatagc tgtggcctga ccgcacactg caacaacttt caatgcccca 5520 atttacaacc ttggtgtgtt gcctcctcac ccctggcaca atgagacttt gatcccatgc 5580 ctaatctggt gtgctctgga cttgcaggat ccctggagtt gcagctacca gacatggtgc 5640 gtggggcccg gggccccgag ctctgctctg tgcagctggc ccgcaatggg gccgggccga 5700 ggtgcaatct gtttcgctgc cgccgcctga ggggctggtg gccggtagtg aagctgaagg 5760 aggcagagga cgtggagcgg gaggcgcagg aggctcaggc tggcaagaag aagcgaaagc 5820 agaggaggag gaagggccgg ccagaagacc tggagttcac agacatgggt ggcaatgtgt 5880 acatcctcac gggcaaggtg gaggcagagt ttgagctgct gactgtggag gaggccgaga 5940 aacggccagt ggggaagggg cggaagcagc cagagcctct ggagaaaccc agccgcccca 6000 aaacttcctt caactggttt gtgaacccgc tgaagacctt tgtcttcttc atctggcgcc 6060 ggtactggcg caccctggtg ctgctgctac tggtgctgct caccgtcttc ctcctcctgg 6120 tcttctacac catccctggc cagatcagcc aggtcatctt ccgtcccctc cacaagtgac 6180 tctcgctgac cttggacact cacccagggt gccaaccctt caatgcctgc tcctggaagt 6240 ctttcttacc catgtgagct accccagagt ctagtgcttc ctctgaataa acctatcaca 6300 gcc 6303

TABLE LIII(c) Nucleotide sequence alignment of 121P1F1 v.1 (SEQ ID NO: 347) and 158P3D2 v.15 (SEQ ID NO: 348)

TABLE LIV(c) Peptide sequences of protein coded by 158P3D2 v.15 (SEQ ID NO: 349) MALTVSVQRL TGLTGTHDRQ VKLTFRGFTQ KTRKIHCGPE ADIGELFRWP HYGAPLAGEC 60 LSVQVVNCSR VFSLRPLGTL VISLQQLQNA GHLVLREALV DENLQVSPIQ VELDLKYQPP 120 EGATGAWSEE DFGAPIQDSF ELIIPNVGFQ ELEPGEAQLE RRAVALGRRL ARSLGQQDDE 180 ENELELELEQ DLDDEPDVEL SGVMFSPLKS RARALAHGDP FQVSRAQDFQ VGVTVLEAQK 240 LVGVNINPYV AVQVGGQRRV TATQRGTSCP FYNEYFLFEF HDTRLRLQDL LLEITVSGVG 300 VTSVLQRRGD EKAAGLTPPS PKAFHSQTLP FMATRIGTFR MDLGIILDQP DGQFYQRWVP 360 LHDPRDTRAG TKGFIKVTLS VRARGDLPPP MLPPAPGHCS DIEKNLLLPR GVPAERPWAR 420 LRVRLYRAEG LPALRLGLLG SLVRALHDQR VLVEPYVRVS FLGQEGETSV SAEAAAPEWN 480 EQLSFVELFP PLTRSLRLQL RDDAPLVDAA LATHVPDLRR ISHPGRAAGF NPTFGPAWVP 540 LYGSPPGAGL RDSLQGLNEG VGQGIWFRGR LLLAVSMQVL EGRAEPEPPQ AQQGSTLSRL 600 TRKKKKKARR DQTPKAVPQH LDASPGAEGP EIPRAMEVEV EELLPLPENV LAPCEDFLLF 660 GVLFEATMID PTVASQPISF EISIGRAGRL EEQLGRGSRA GEGTEGAAVE AQPLLGARPE 720 EEKEEEELGT HAQRPEPMDG SGPYFCLPLC HCKPCMHVWS CWEDHTWRLQ SSNCVRKVAE 780 RLDQGLQEVE RLQRKPGPGA CAQLKQALEV LVAGSRQFCH GAERRTMTRP NALDRCRGKL 840 LVHSLNLLAK QGLRLLRGLR RRNVQKKVAL AKKLLAKLRF LAEEHNFDED EMDDPGDSDG 900 VNLISMVGEI QDQGEAEVKG TVSPKKAVAT LKIYNRSLKE EFNHFEDWLN VFPLYRGQGG 960 QDGGGEEEGS GHLVGKFKGS FLIYPESEAV LFSEPQISRG IPQNRPIKLL VRVYVVKLRN 1020 LCKIQGHEDF CLFSAATNLA PADPNGKADP YVVVSAGRER QDTKERYIPK QLNPIFGEIL 1080 ELSISLPAET ELTVAVFDHD LVGSDDLIGE THIDLENRFY SHHRANCGLA SQYEVWVQQG 1140 PQEPF 1145

TABLE LV(c) Amino acid sequence alignment of 121P1F1 v.1 and 158P3D2 v.15 No significant similarity

TABLE LII(d) Nucleotide sequence of transcript variant 158P3D2 v.16 (SEQ ID NO: 350) caggtgggcg ggctggtggg cagaagggca gacgggcaga ggaagtgcca gtgccactgg 60 gaccatggct ctgacggtaa gcgtgcaacg actaacaggg ctgaccggca cccacgaccg 120 acaagtgaag ctcacctttc gaggctttac ccagaaaaca agaaaaattc actgtggtcc 180 agaagcagat atcggtgagc tgttccgatg gccccactat ggggctccac tggctgggga 240 gtgtctgtct gtgcaggtgg tcaactgcag ccgtgtattc agccttaggc ctctagggac 300 cctggtgatc tccctgcagc agctacagaa tgctgggcat ttggtgctac gggaagccct 360 agtggatgag aatcttcaag tgtccccgat ccaggtggag cttgacctga agtaccagcc 420 cccagagggc gctactggag cctggtcaga ggaggacttt ggggcaccca tccaggacag 480 cttcgagtta atcatcccca atgtgggctt ccaggaactg gagcctgggg aggcccagct 540 ggagcggcgg gcagtggctc taggccgcag gctagctcga agtctaggcc agcaggacga 600 tgaagagaat gagctggagc ttgagctgga gcaggacctg gatgatgagc ctgacgtgga 660 actttctggt gttatgttca gccccctcaa gagccgcgcc agggccctgg cccatgggga 720 tcccttccag gtgtccagag ctcaagactt ccaggtggga gtcactgtgc tggaagccca 780 gaaactggtg ggagtcaaca ttaaccccta tgtggccgtg caagtggggg ggcagcgccg 840 tgtgaccgcc acacagcgtg ggaccagttg ccccttctac aatgagtact tcttgttcga 900 atttcatgac acgcggcttc gtctccaaga cttgctgctg gagatcacgg tgagtggggt 960 aggggtgacc agtgtccttc agagaagggg ggatgagaaa gctgcaggac taacaccacc 1020 ttcccccaag gctttccatt cgcagaccct cccctttatg gccacccgga taggcacctt 1080 caggatggac ctgggcatca tcttggacca gccagatggc cagttctacc aaagatgggt 1140 tccgctgcat gatccccgag acacccgcgc cgggaccaag ggtttcatta aggtcacctt 1200 gtccgtgagg gcgcgcgggg acctgccccc tccaatgcta cccccggccc cagggcactg 1260 ttcggacatc gagaagaacc tgctcctgcc gcgcggggtg cccgccgaga ggccatgggc 1320 gcggctccgc gtgcgcctgt accgcgccga ggggcttccc gcgctgcgcc tggggctgct 1380 gggcagcctg gtccgcgccc tgcacgacca gcgcgtcctg gtggagccct atgtgcgggt 1440 gtctttcctg gggcaggagg gcgagacgtc ggtgagcgcc gaggcggcgg cgcccgaatg 1500 gaacgagcag ctgagcttcg tggagctctt cccgccgctg acgcgcagcc tccgcctgca 1560 gctgcgggac gacgcgcccc tggtcgacgc ggcactcgct acgcacgtgc cggacctgag 1620 gcggatctcc catccgggcc gcgcggcggg gtttaaccct accttcggcc cggcctgggt 1680 gcccctctat ggctcgcccc ccggcgcggg gctccgggat agtcttcaag gtctcaacga 1740 aggcgttggc caaggcattt ggttccgcgg ccgccttctg ctggctgtgt ccatgcaggt 1800 gttggaaggg agagctgaac ctgagcctcc ccaggcccag caggggtcca cgttgtcccg 1860 gctcacccga aagaagaaaa agaaagccag aagggatcag accccaaagg cggttccgca 1920 gcacttggac gccagccccg gtgccgaggg gcctgagatc ccccgtgcca tggaggtgga 1980 ggtggaggag ctgctgccgc tgccagagaa tgtcctggcg ccctgtgaag atttcctgct 2040 tttcggtgtg ctcttcgagg ccaccatgat cgaccccacc gtggcctccc agcccatcag 2100 cttcgagatc tccattggtc gcgcaggccg tctggaggag caattgggcc gagggtccag 2160 ggctggggag ggaactgagg gtgcagccgt ggaggctcag cctctgctgg gagccaggcc 2220 agaggaggag aaagaggagg aagaactggg gacccatgct cagcggcctg agcccatgga 2280 cggcagtggg ccatacttct gcttgcccct ctgtcactgc aagccatgca tgcatgtgtg 2340 gagttgctgg gaggaccaca cctggcgcct gcagagcagc aactgcgtgc gcaaagtggc 2400 cgagaggctg gaccaggggc tgcaggaggt tgagagactg cagcgcaagc cggggcctgg 2460 cgcctgtgca cagctcaagc aggcactgga agtactggtg gctgggagca gacagttttg 2520 ccacggtgcc gagcgcagga cgatgacccg gcccaatgcc ctggatcgat gccgagggaa 2580 actcctggtg cacagcctga accttttggc taagcaagga ctgcgacttc tacgcggcct 2640 gagacggcgc aatgtgcaaa agaaggtggc actggccaag aagctcctgg caaaactgcg 2700 ctttctggct gaggagcccc agccacccct ccccgatgtg ctggtctgga tgctcagcgg 2760 gcagcgccgt gtggcctggg cccggatccc tgcccaggat gtgctgttct ctgtggttga 2820 ggaggaacgg ggccgagact gtgggaagat ccagagtcta atgctcacgg cacccggggc 2880 agcccctggt gaggtctgtg ccaagctgga gctcttcctg cggctgggcc tgggcaagca 2940 agccaaggcc tgcacctctg agctgccccc ggatttgctg cccgagccct cagccgggct 3000 gccctccagc ctacaccggg acgactttag ctacttccaa ctccgggctc acttgtacca 3060 ggcccggggt gtgttggctg cagatgacag tggcctctcg gacccctttg ctcgagtcct 3120 catctctacc cagtgtcaga ccacacgggt cctggagcag acgctgagcc ctctgtggga 3180 tgaactcctg gtatttgagc agttgatcgt ggatgggagg agggagcacc tgcaggagga 3240 gcctccatta gtgatcatca atgtatttga ccacaataag tttggccccc ccgtgttcct 3300 gggcagggca ctggccgccc caagggtaaa gctgatggag gacccatacc aacgcccaga 3360 gttgcagttc ttccccctga ggaagggacc ctgggcagcc ggagagctca ttgccgcctt 3420 tcaactcatt gaactagact acagtggccg acttgagccc tcagtgccca gtgaggtgga 3480 gccccaggat ctggcacccc tggttgagcc ccactctgga cgcctgtccc ttccacccaa 3540 cgtgtgccca gtgctcaggg agttccgtgt tgaggtgctg ttctggggtc ttaggggact 3600 tggtcgtgtg catctgctcg aggtggagca gccccaggtt gtactggagg tggctgggca 3660 aggtgtggag tctgaggtcc tggccagcta ccgtgagagc cccaatttca ctgagcttgt 3720 caggcatctg acagtggtct tcaaagacac agctcctctc ttccaccccc aggacttgcc 3780 ggagcagcct tacttgcagc ctccactcag catcttggtg attgagcgcc gggcctttgg 3840 ccacacagtc cttgtgggtt cccacattgt cccccacatg ctgcgattca catttcgggg 3900 tcatgaggat cctcctgagg aggaaggaga gatggaggag acaggggata tgatgcccaa 3960 gggacctcaa ggacagaagt ccctggatcc cttcttggct gaagcgggta tatccagaca 4020 gctcctgaag cctcctctga agaagctccc actaggaggc ctcctaaatc aaggccctgg 4080 gctggaggaa gacatcccag atccagagga gctcgactgg gggtccaagt actatgcgtc 4140 gctgcaggag ctccaggggc agcacaactt tgatgaagat gaaatggatg atcctggaga 4200 ttcagatggg gtcaacctca tttctatggt tggggagatc caagaccagg gtgaggctga 4260 agtcaaaggc actgtgtccc caaaaaaagc agttgccacc ctgaagatct acaacaggtc 4320 cctgaaggaa gaatttaacc actttgaaga ctggctgaat gtgtttcctc tgtaccgagg 4380 gcaagggggc caggatggag gtggagaaga ggaaggatct ggacaccttg tgggcaagtt 4440 caagggctcc ttcctcattt accctgaatc agaggcagtg ttgttctctg agccccagat 4500 ctcccggggg atcccacaga accggcccat caagctcctg gtcagagtgt atgttgtaaa 4560 ggctaccaac ctggctcctg cagaccccaa tggcaaagca gacccttacg tggtggtgag 4620 cgctggccgg gagcggcagg acaccaagga acgctacatc cccaagcagc tcaaccccat 4680 ctttggagag atcctggagc taagcatctc tctcccagct gagacggagc tgacggtcgc 4740 cgtatttgat catgacctcg tgggttctga cgacctcatc ggggagaccc acattgatct 4800 ggaaaaccga ttctatagcc accacagagc aaactgtggg ctggcctccc agtatgaagt 4860 agatggttac aatgcctggc gtgatgcatt ctggccttcg cagatcctgg cggggctgtg 4920 ccaacgctgt ggcctccctg cccctgaata ccgagccggt gctgtcaagg tgggcagcaa 4980 agtcttcctg acaccaccgg agaccctgcc cccagtggcg agcggggacc ctgaagaggc 5040 ccaggcattg cttgtgctgc ggcgctggca ggaaatgccg ggttttggga tccagctggt 5100 acccgagcat gtagaaacca ggcctctcta ccatccccac agcccagggc tgctacaggg 5160 atctcttcac atgtggattg acatctttcc tcaagatgtg cctgctccac ccccagttga 5220 catcaagcct cggcagccaa tcagctatga gctcagagtt gtcatctgga acacggagga 5280 tgtggttctg gatgacgaga atccactcac cggagagatg tcgagtgaca tctatgtgaa 5340 gagctgggtg aaggggttgg agcatgacaa gcaggagaca gacgttcact tcaactccct 5400 gactggggag gggaacttca attggcgctt tgtgttccgc tttgactacc tgcccacgga 5460 gcgggaggtg agcgtctggc gcaggtctgg accctttgcc ctggaggagg cggagttccg 5520 gcagcctgca gtgctggtcc tgcaggtctg ggactatgac cgcatctctg ccaatgactt 5580 ccttggatcc ctggagttgc agctaccaga catggtgcgt ggggcccggg gccccgagct 5640 ctgctctgtg cagctggccc gcaatggggc cgggccgagg tgcaatctgt ttcgctgccg 5700 ccgcctgagg ggctggtggc cggtagtgaa gctgaaggag gcagaggacg gcaaggtgga 5760 ggcagagttt gagctgctga ctgtggagga ggccgagaaa cggccagtgg ggaaggggcg 5820 gaagcagcca gagcctctgg agaaacccag ccgccccaaa acttccttca actggtttgt 5880 gaacccgctg aagacctttg tcttcttcat ctggcgccgg tactggcgca ccctggtgct 5940 gctgctactg gtgctgctca ccgtcttcct cctcctggtc ttctacacca tccctggcca 6000 gatcagccag gtcatcttcc gtcccctcca caagtgactc tcgctgacct tggacactca 6060 cccagggtgc caacccttca atgcctgctc ctggaagtct ttcttaccca tgtgagctac 6120 cccagagtcta gtgcttcct ctgaataaacctatcacagc c 6161

TABLE LIII(d) Nucleotide sequence alignment of 121P1F1 v.1 (SEQ ID NO: 351) and 158P3D2 v.16 (SEQ ID NO: 352)

TABLE LIV(d) Peptide sequences of protein coded by 158P3D2 v.16 (SEQ ID NO: 353) MALTVSVQRL TGLTGTHDRQ VKLTFRGFTQ KTRKIHCGPE ADIGELFRWP HYGAPLAGEC 60 LSVQVVNCSR VFSLRPLGTL VISLQQLQNA GHLVLREALV DENLQVSPIQ VELDLKYQPP 120 EGATGAWSEE DFGAPIQDSF ELIIPNVGFQ ELEPGEAQLE RRAVALGRRL ARSLGQQDDE 180 ENELELELEQ DLDDEPDVEL SGVMFSPLKS RARALAHGDP FQVSRAQDFQ VGVTVLEAQK 240 LVGVNINPYV AVQVGGQRRV TATQRGTSCP FYNEYFLFEF HDTRLRLQDL LLEITVSGVG 300 VTSVLQRRGD EKAAGLTPPS PKAFHSQTLP FMATRIGTFR MDLGIILDQP DGQFYQRWVP 360 LHDPRDTRAG TKGFIKVTLS VRARGDLPPP MLPPAPGHCS DIEKNLLLPR GVPAERPWAR 420 LRVRLYRAEG LPALRLGLLG SLVRALHDQR VLVEPYVRVS FLGQEGETSV SAEAAAPEWN 480 EQLSFVELFP PLTRSLRLQL RDDAPLVDAA LATHVPDLRR ISHPGRAAGF NPTFGPAWVP 540 LYGSPPGAGL RDSLQGLNEG VGQGIWFRGR LLLAVSMQVL EGRAEPEPPQ AQQGSTLSRL 600 TRKKKKKARR DQTPKAVPQH LDASPGAEGP EIPRAMEVEV EELLPLPENV LAPCEDFLLF 660 GVLFEATMID PTVASQPISF EISIGRAGRL EEQLGRGSRA GEGTEGAAVE AQPLLGARPE 720 EEKEEEELGT HAQRPEPMDG SGPYFCLPLC HCKPCMHVWS CWEDHTWRLQ SSNCVRKVAE 780 RLDQGLQEVE RLQRKPGPGA CAQLKQALEV LVAGSRQFCH GAERRTMTRP NALDRCRGKL 840 LVHSLNLLAK QGLRLLRGLR RRNVQKKVAL AKKLLAKLRF LAEEPQPPLP DVLVWMLSGQ 900 RRVAWARIPA QDVLFSVVEE ERGRDCGKIQ SLMLTAPGAA PGEVCAKLEL FLRLGLGKQA 960 KACTSELPPD LLPEPSAGLP SSLHRDDFSY FQLRAHLYQA RGVLAADDSG LSDPFARVLI 1020 STQCQTTRVL EQTLSPLWDE LLVFEQLIVD GRREHLQEEP PLVIINVFDH NKFGPPVFLG 1080 RALAAPRVKL MEDPYQRPEL QFFPLRKGPW AAGELIAAFQ LIELDYSGRL EPSVPSEVEP 1140 QDLAPLVEPH SGRLSLPPNV CPVLREFRVE VLFWGLRGLG RVHLLEVEQP QVVLEVAGQG 1200 VESEVLASYR ESPNFTELVR HLTVVFKDTA PLFHPQDLPE QPYLQPPLSI LVIERRAFGH 1260 TVLVGSHIVP HMLRFTFRGH EDPPEEEGEM EETGDMMPKG PQGQKSLDPF LAEAGISRQL 1320 LKPPLKKLPL GGLLNQGPGL EEDIPDPEEL DWGSKYYASL QELQGQHNFD EDEMDDPGDS 1380 DGVNLISMVG EIQDQGEAEV KGTVSPKKAV ATLKIYNRSL KEEFNHFEDW LNVFPLYRGQ 1440 GGQDGGGEEE GSGHLVGKFK GSFLIYPESE AVLFSEPQIS RGIPQNRPIK LLVRVYVVKA 1500 TNLAPADPNG KADPYVVVSA GRERQDTKER YIPKQLNPIF GEILELSISL PAETELTVAV 1560 FDHDLVGSDD LIGETHIDLE NRFYSHHRAN CGLASQYEVD GYNAWRDAFW PSQILAGLCQ 1620 RCGLPAPEYR AGAVKVGSKV FLTPPETLPP VASGDPEEAQ ALLVLRRWQE MPGFGIQLVP 1680 EHVETRPLYH PHSPGLLQGS LHMWIDIFPQ DVPAPPPVDI KPRQPISYEL RVVIWNTEDV 1740 VLDDENPLTG EMSSDIYVKS WVKGLEHDKQ ETDVHFNSLT GEGNFNWRFV FRFDYLPTER 1800 EVSVWRRSGP FALEEAEFRQ PAVLVLQVWD YDRISANDFL GSLELQLPDM VRGARGPELC 1860 SVQLARNGAG PRCNLFRCRR LRGWWPVVKL KEAEDGKVEA EFELLTVEEA EKRPVGKGRK 1920 QPEPLEKPSR PKTSFNWFVN PLKTFVFFIW RRYWRTLVLL LLVLLTVFLL LVFYTIPGQI 1980 SQVIFRPLHK 1990

TABLE LV(d) Amino acid sequence alignment of 121P1F1 v.1 (SEQ ID NO: 354) and 158P3D2 v.16 (SEQ ID NO: 355) Score = 580 bits (1496), Expect = e-164Identities = 288/328 (87%), Positives = 288/328 (87%), Gaps = 40/328 (12%) V.1: 1 MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV 60 MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV V.16: 1703 MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV 1762 V.1: 61 KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA 120 KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA V.16: 1763 KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA 1822 V.1: 121 VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR 180 VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR V.16: 1823 VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR 1882 V.1: 181 GWWPVVKLKEAEDVEREAQEAQAGKKKRKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEF 240 GWWPVVKLKEAED                                        GKVEAEF V.16: 1883 GWWPVVKLKEAED----------------------------------------GKVEAEF 1902 V.1: 241 ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL 300 ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL V.16: 1903 ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL 1962 V.1: 301 VLLTVFLLLVFYTIPGQISQVIFRPLHK 328 VLLTVFLLLVFYTIPGQISQVIFRPLHK V.16: 1963 VLLTVFLLLVFYTIPGQISQVIFRPLHK 1990

TABLE LII(e) Nucleotide sequence of transcript variant 158P3D2 v.17 (SEQ ID NO: 356) caggtgggcg ggctggtggg cagaagggca gacgggcaga ggaagtgcca gtgccactgg 60 gaccatggct ctgacggtaa gcgtgcaacg actaacaggg ctgaccggca cccacgaccg 120 acaagtgaag ctcacctttc gaggctttac ccagaaaaca agaaaaattc actgtggtcc 180 agaagcagat atcggtgagc tgttccgatg gccccactat ggggctccac tggctgggga 240 gtgtctgtct gtgcaggtgg tcaactgcag ccgtgtattc agccttaggc ctctagggac 300 cctggtgatc tccctgcagc agctacagaa tgctgggcat ttggtgctac gggaagccct 360 agtggatgag aatcttcaag tgtccccgat ccaggtggag cttgacctga agtaccagcc 420 cccagagggc gctactggag cctggtcaga ggaggacttt ggggcaccca tccaggacag 480 cttcgagtta atcatcccca atgtgggctt ccaggaactg gagcctgggg aggcccagct 540 ggagcggcgg gcagtggctc taggccgcag gctagctcga agtctaggcc agcaggacga 600 tgaagagaat gagctggagc ttgagctgga gcaggacctg gatgatgagc ctgacgtgga 660 actttctggt gttatgttca gccccctcaa gagccgcgcc agggccctgg cccatgggga 720 tcccttccag gtgtccagag ctcaagactt ccaggtggga gtcactgtgc tggaagccca 780 gaaactggtg ggagtcaaca ttaaccccta tgtggccgtg caagtggggg ggcagcgccg 840 tgtgaccgcc acacagcgtg ggaccagttg ccccttctac aatgagtact tcttgttcga 900 atttcatgac acgcggcttc gtctccaaga cttgctgctg gagatcacgg tgagtggggt 960 aggggtgacc agtgtccttc agagaagggg ggatgagaaa gctgcaggac taacaccacc 1020 ttcccccaag gctttccatt cgcagaccct cccctttatg gccacccgga taggcacctt 1080 caggatggac ctgggcatca tcttggacca gccagatggc cagttctacc aaagatgggt 1140 tccgctgcat gatccccgag acacccgcgc cgggaccaag ggtttcatta aggtcacctt 1200 gtccgtgagg gcgcgcgggg acctgccccc tccaatgcta cccccggccc cagggcactg 1260 ttcggacatc gagaagaacc tgctcctgcc gcgcggggtg cccgccgaga ggccatgggc 1320 gcggctccgc gtgcgcctgt accgcgccga ggggcttccc gcgctgcgcc tggggctgct 1380 gggcagcctg gtccgcgccc tgcacgacca gcgcgtcctg gtggagccct atgtgcgggt 1440 gtctttcctg gggcaggagg gcgagacgtc ggtgagcgcc gaggcggcgg cgcccgaatg 1500 gaacgagcag ctgagcttcg tggagctctt cccgccgctg acgcgcagcc tccgcctgca 1560 gctgcgggac gacgcgcccc tggtcgacgc ggcactcgct acgcacgtgc cggacctgag 1620 gcggatctcc catccgggcc gcgcggcggg gtttaaccct accttcggcc cggcctgggt 1680 gcccctctat ggctcgcccc ccggcgcggg gctccgggat agtcttcaag gtctcaacga 1740 aggcgttggc caaggcattt ggttccgcgg ccgccttctg ctggctgtgt ccatgcaggt 1800 gttggaaggg agagctgaac ctgagcctcc ccaggcccag caggggtcca cgttgtcccg 1860 gctcacccga aagaagaaaa agaaagccag aagggatcag accccaaagg cggttccgca 1920 gcacttggac gccagccccg gtgccgaggg gcctgagatc ccccgtgcca tggaggtgga 1980 ggtggaggag ctgctgccgc tgccagagaa tgtcctggcg ccctgtgaag atttcctgct 2040 tttcggtgtg ctcttcgagg ccaccatgat cgaccccacc gtggcctccc agcccatcag 2100 cttcgagatc tccattggtc gcgcaggccg tctggaggag caattgggcc gagggtccag 2160 ggctggggag ggaactgagg gtgcagccgt ggaggctcag cctctgctgg gagccaggcc 2220 agaggaggag aaagaggagg aagaactggg gacccatgct cagcggcctg agcccatgga 2280 cggcagtggg ccatacttct gcttgcccct ctgtcactgc aagccatgca tgcatgtgtg 2340 gagttgctgg gaggaccaca cctggcgcct gcagagcagc aactgcgtgc gcaaagtggc 2400 cgagaggctg gaccaggggc tgcaggaggt tgagagactg cagcgcaagc cggggcctgg 2460 cgcctgtgca cagctcaagc aggcactgga agtactggtg gctgggagca gacagttttg 2520 ccacggtgcc gagcgcagga cgatgacccg gcccaatgcc ctggatcgat gccgagggaa 2580 actcctggtg cacagcctga accttttggc taagcaagga ctgcgacttc tacgcggcct 2640 gagacggcgc aatgtgcaaa agaaggtggc actggccaag aagctcctgg caaaactgcg 2700 ctttctggct gaggagcccc agccacccct ccccgatgtg ctggtctgga tgctcagcgg 2760 gcagcgccgt gtggcctggg cccggatccc tgcccaggat gtgctgttct ctgtggttga 2820 ggaggaacgg ggccgagact gtgggaagat ccagagtcta atgctcacgg cacccggggc 2880 agcccctggt gaggtctgtg ccaagctgga gctcttcctg cggctgggcc tgggcaagca 2940 agccaaggcc tgcacctctg agctgccccc ggatttgctg cccgagccct cagccgggct 3000 gccctccagc ctacaccggg acgactttag ctacttccaa ctccgggctc acttgtacca 3060 ggcccggggt gtgttggctg cagatgacag tggcctctcg gacccctttg ctcgagtcct 3120 catctctacc cagtgtcaga ccacacgggt cctggagcag acgctgagcc ctctgtggga 3180 tgaactcctg gtatttgagc agttgatcgt ggatgggagg agggagcacc tgcaggagga 3240 gcctccatta gtgatcatca atgtatttga ccacaataag tttggccccc ccgtgttcct 3300 gggcagggca ctggccgccc caagggtaaa gctgatggag gacccatacc aacgcccaga 3360 gttgcagttc ttccccctga ggaagggacc ctgggcagcc ggagagctca ttgccgcctt 3420 tcaactcatt gaactagact acagtggccg acttgagccc tcagtgccca gtgaggtgga 3480 gccccaggat ctggcacccc tggttgagcc ccactctgga cgcctgtccc ttccacccaa 3540 cgtgtgccca gtgctcaggg agttccgtgt tgaggtgctg ttctggggtc ttaggggact 3600 tggtcgtgtg catctgctcg aggtggagca gccccaggtt gtactggagg tggctgggca 3660 aggtgtggag tctgaggtcc tggccagcta ccgtgagagc cccaatttca ctgagcttgt 3720 caggcatctg acagtggtct tcaaagacac agctcctctc ttccaccccc aggacttgcc 3780 ggagcagcct tacttgcagc ctccactcag catcttggtg attgagcgcc gggcctttgg 3840 ccacacagtc cttgtgggtt cccacattgt cccccacatg ctgcgattca catttcgggg 3900 tcatgaggat cctcctgagg aggaaggaga gatggaggag acaggggata tgatgcccaa 3960 gggacctcaa ggacagaagt ccctggatcc cttcttggct gaagcgggta tatccagaca 4020 gctcctgaag cctcctctga agaagctccc actaggaggc ctcctaaatc aaggccctgg 4080 gctggaggaa gacatcccag atccagagga gctcgactgg gggtccaagt actatgcgtc 4140 gctgcaggag ctccaggggc agcacaactt tgatgaagat gaaatggatg atcctggaga 4200 ttcagatggg gtcaacctca tttctatggt tggggagatc caagaccagg gtgaggctga 4260 agtcaaaggc actgtgtccc caaaaaaagc agttgccacc ctgaagatct acaacaggtc 4320 cctgaaggaa gaatttaacc actttgaaga ctggctgaat gtgtttcctc tgtaccgagg 4380 gcaagggggc caggatggag gtggagaaga ggaaggatct ggacaccttg tgggcaagtt 4440 caagggctcc ttcctcattt accctgaatc agaggcagtg ttgttctctg agccccagat 4500 ctcccggggg atcccacaga accggcccat caagctcctg gtcagagtgt atgttgtaaa 4560 ggctaccaac ctggctcctg cagaccccaa tggcaaagca gacccttacg tggtggtgag 4620 cgctggccgg gagcggcagg acaccaagga acgctacatc cccaagcagc tcaaccccat 4680 ctttggagag atcctggagc taagcatctc tctcccagct gagacggagc tgacggtcgc 4740 cgtatttgat catgacctcg tgggttctga cgacctcatc ggggagaccc acattgatct 4800 ggaaaaccga ttctatagcc accacagagc aaactgtggg ctggcctccc agtatgaagt 4860 agatggttac aatgcctggc gtgatgcatt ctggccttcg cagatcctgg cggggctgtg 4920 ccaacgctgt ggcctccctg cccctgaata ccgagccggt gctgtcaagg tgggcagcaa 4980 agtcttcctg acaccaccgg agaccctgcc cccaggcagc agcagcccca cagtggcgag 5040 cggggaccct gaagaggccc aggcattgct tgtgctgcgg cgctggcagg aaatgccggg 5100 ttttgggatc cagctggtac ccgagcatgt agaaaccagg cctctctacc atccccacag 5160 cccagggctg ctacagggat ctcttcacat gtggattgac atctttcctc aagatgtgcc 5220 tgctccaccc ccagttgaca tcaagcctcg gcagccaatc agctatgagc tcagagttgt 5280 catctggaac acggaggatg tggttctgga tgacgagaat ccactcaccg gagagatgtc 5340 gagtgacatc tatgtgaaga gctgggtgaa ggggttggag catgacaagc aggagacaga 5400 cgttcacttc aactccctga ctggggaggg gaacttcaat tggcgctttg tgttccgctt 5460 tgactacctg cccacggagc gggaggtgag cgtctggcgc aggtctggac cctttgccct 5520 ggaggaggcg gagttccggc agcctgcagt gctggtcctg caggtctggg actatgaccg 5580 catctctgcc aatgacttcc ttggatccct ggagttgcag ctaccagaca tggtgcgtgg 5640 ggcccggggc cccgagctct gctctgtgca gctggcccgc aatggggccg ggccgaggtg 5700 caatctgttt cgctgccgcc gcctgagggg ctggtggccg gtagtgaagc tgaaggaggc 5760 agaggacgtg gagcgggagg cgcaggaggc tcaggctggc aagaagaagc gaaagcagag 5820 gaggaggaag ggccggccag aagacctgga gttcacagac atgggtggca atgtgtacat 5880 cctcacgggc aaggtggagg cagagtttga gctgctgact gtggaggagg ccgagaaacg 5940 gccagtgggg aaggggcgga agcagccaga gcctctggag aaacccagcc gccccaaaac 6000 ttccttcaac tggtttgtga acccgctgaa gacctttgtc ttcttcatct ggcgccggta 6060 ctggcgcacc ctggtgctgc tgctactggt gctgctcacc gtcttcctcc tcctggtctt 6120 ctacaccatc cctggccaga tcagccaggt catcttccgt cccctccaca agtgactctc 6180 gctgaccttg gacactcacc cagggtgcca acccttcaat gcctgctcct ggaagtcttt 6240 cttacccatg tgagctaccc cagagtctag tgcttcctct gaataaacct atcacagcc 6299

TABLE LIII(e) Nucleotide sequence alignment of 121P1F1 v.1 (SEQ ID NO: 357) and 158P3D2 v.17 (SEQ ID NO: 358)

TABLE LIV(e) Peptide sequences of protein coded by 158P3D2 v.17 (SEQ ID NO: 359) MALTVSVQRL TGLTGTHDRQ VKLTFRGFTQ KTRKIHCGPE ADIGELFRWP HYGAPLAGEC 60 LSVQVVNCSR VFSLRPLGTL VISLQQLQNA GHLVLREALV DENLQVSPIQ VELDLKYQPP 120 EGATGAWSEE DFGAPIQDSF ELIIPNVGFQ ELEPGEAQLE RRAVALGRRL ARSLGQQDDE 180 ENELELELEQ DLDDEPDVEL SGVMFSPLKS RARALAHGDP FQVSRAQDFQ VGVTVLEAQK 240 LVGVNINPYV AVQVGGQRRV TATQRGTSCP FYNEYFLFEF HDTRLRLQDL LLEITVSGVG 300 VTSVLQRRGD EKAAGLTPPS PKAFHSQTLP FMATRIGTFR MDLGIILDQP DGQFYQRWVP 360 LHDPRDTRAG TKGFIKVTLS VRARGDLPPP MLPPAPGHCS DIEKNLLLPR GVPAERPWAR 420 LRVRLYRAEG LPALRLGLLG SLVRALHDQR VLVEPYVRVS FLGQEGETSV SAEAAAPEWN 480 EQLSFVELFP PLTRSLRLQL RDDAPLVDAA LATHVPDLRR ISHPGRAAGF NPTFGPAWVP 540 LYGSPPGAGL RDSLQGLNEG VGQGIWFRGR LLLAVSMQVL EGRAEPEPPQ AQQGSTLSRL 600 TRKKKKKARR DQTPKAVPQH LDASPGAEGP EIPRAMEVEV EELLPLPENV LAPCEDFLLF 660 GVLFEATMID PTVASQPISF EISIGRAGRL EEQLGRGSRA GEGTEGAAVE AQPLLGARPE 720 EEKEEEELGT HAQRPEPMDG SGPYFCLPLC HCKPCMHVWS CWEDHTWRLQ SSNCVRKVAE 780 RLDQGLQEVE RLQRKPGPGA CAQLKQALEV LVAGSRQFCH GAERRTMTRP NALDRCRGKL 840 LVHSLNLLAK QGLRLLRGLR RRNVQKKVAL AKKLLAKLRF LAEEPQPPLP DVLVWMLSGQ 900 RRVAWARIPA QDVLFSVVEE ERGRDCGKIQ SLMLTAPGAA PGEVCAKLEL FLRLGLGKQA 960 KACTSELPPD LLPEPSAGLP SSLHRDDFSY FQLRAHLYQA RGVLAADDSG LSDPFARVLI 1020 STQCQTTRVL EQTLSPLWDE LLVFEQLIVD GRREHLQEEP PLVIINVFDH NKFGPPVFLG 1080 RALAAPRVKL MEDPYQRPEL QFFPLRKGPW AAGELIAAFQ LIELDYSGRL EPSVPSEVEP 1140 QDLAPLVEPH SGRLSLPPNV CPVLREFRVE VLFWGLRGLG RVHLLEVEQP QVVLEVAGQG 1200 VESEVLASYR ESPNFTELVR HLTVVFKDTA PLFHPQDLPE QPYLQPPLSI LVIERRAFGH 1260 TVLVGSHIVP HMLRFTFRGH EDPPEEEGEM EETGDMMPKG PQGQKSLDPF LAEAGISRQL 1320 LKPPLKKLPL GGLLNQGPGL EEDIPDPEEL DWGSKYYASL QELQGQHNFD EDEMDDPGDS 1380 DGVNLISMVG EIQDQGEAEV KGTVSPKKAV ATLKIYNRSL KEEFNHFEDW LNVFPLYRGQ 1440 GGQDGGGEEE GSGHLVGKFK GSFLIYPESE AVLFSEPQIS RGIPQNRPIK LLVRVYVVKA 1500 TNLAPADPNG KADPYVVVSA GRERQDTKER YIPKQLNPIF GEILELSISL PAETELTVAV 1560 FDHDLVGSDD LIGETHIDLE NRFYSHHRAN CGLASQYEVD GYNAWRDAFW PSQILAGLCQ 1620 RCGLPAPEYR AGAVKVGSKV FLTPPETLPP GSSSPTVASG DPEEAQALLV LRRWQEMPGF 1680 GIQLVPEHVE TRPLYHPHSP GLLQGSLHMW IDIFPQDVPA PPPVDIKPRQ PISYELRVVI 1740 WNTEDVVLDD ENPLTGEMSS DIYVKSWVKG LEHDKQETDV HFNSLTGEGN FNWRFVFRFD 1800 YLPTEREVSV WRRSGPFALE EAEFRQPAVL VLQVWDYDRI SANDFLGSLE LQLPDMVRGA 1860 RGPELCSVQL ARNGAGPRCN LFRCRRLRGW WPVVKLKEAE DVEREAQEAQ AGKKKRKQRR 1920 RKGRPEDLEF TDMGGNVYIL TGKVEAEFEL LTVEEAEKRP VGKGRKQPEP LEKPSRPKTS 1980 FNWFVNPLKT FVFFIWRRYW RTLVLLLLVL LTVFLLLVFY TIPGQISQVI FRPLHK 2036

TABLE LV(e) Amino acid sequence alignment of 121P1F1 v.1 (SEQ ID NO: 360) and 158P3D2 v.17 (SEQ ID NO: 361) Score = 679 bits (1751), Expect = 0.0Identities = 328/328 (100%), Positives = 328/328 (100%) V.1: 1 MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV 60 MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV v.17: 1709 MWIDIFPQDVPAPPPVDIKPRQPISYELRVVIWNTEDVVLDDENPLTGEMSSDIYVKSWV 1768 V.1: 61 KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA 120 KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA v.17: 1769 KGLEHDKQETDVHFNSLTGEGNFNWRFVFRFDYLPTEREVSVWRRSGPFALEEAEFRQPA 1828 V.1: 121 VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR 180 VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR v.17: 1829 VLVLQVWDYDRISANDFLGSLELQLPDMVRGARGPELCSVQLARNGAGPRCNLFRCRRLR 1888 V.1: 181 GWWPVVKLKEAEDVEREAQEAQAGKKKRKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEF 240 GWWPVVKLKEAEDVEREAQEAQAGKKKRKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEF v.17: 1889 GWWPVVKLKEAEDVEREAQEAQAGKKKRKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEF 1948 V.1: 241 ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL 300 ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL v.17: 1949 ELLTVEEAEKRPVGKGRKQPEPLEKPSRPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLL 2008 V.1: 301 VLLTVFLLLVFYTIPGQISQVIFRPLHK 328 VLLTVFLLLVFYTIPGQISQVIFRPLHK v.17: 2009  VLLTVFLLLVFYTIPGQISQVIFRPLHK 2036

TABLE LII(f) Nucleotide sequence of transcript variant 158P3D2 v.18 (SEQ ID NO: 362) agaagaaagc tggtaggggc tgggagaggg taccacaggg gagtatgatc tacttggggg 60 ccagagaagg ttccctgagg aaatagtacc tgaacttaga cttgaaggat aacagatgtt 120 aactgggagg agagaatgtt ccaggcagag gaaaaggcat atgcaaaagt ccagcgcctt 180 gaaggagcac agctggggtg cctggagtga gatggagctg gaaagatcca ggtggagctt 240 gacctgaagt accagccccc agagggcgct actggagcct ggtcagagga ggactttggg 300 gcacccatcc aggacagctt cgagttaatc atccccaatg tgggcttcca ggaactggag 360 cctggggagg cccagctgga gcggcgggca gtggctctag gccgcaggct agctcgaagt 420 ctaggccagc aggacgatga agagaatgag ctggagcttg agctggagca ggacctggat 480 gatgagcctg acgtggaact ttctggtgtt atgttcagcc ccctcaagag ccgcgccagg 540 gccctggccc atggggatcc cttccaggtg tccagagctc aagacttcca ggtgggagtc 600 actgtgctgg aagcccagaa actggtggga gtcaacatta acccctatgt ggccgtgcaa 660 gtgggggggc agcgccgtgt gaccgccaca cagcgtggga ccagttgccc cttctacaat 720 gagtacttct tgttcgaatt tcatgacacg cggcttcgtc tccaagactt gctgctggag 780 atcacggtga gtggggtagg ggtgaccagt gtccttcaga gaagggggga tgagaaagct 840 gcaggactaa caccaccttc ccccaaggct ttccattcgc agaccctccc ctttatggcc 900 acccggatag gcaccttcag gatggacctg ggcatcatct tggaccagcc aggtatggaa 960 tcgtcccctt attgagactc tgcacggaca agggccctag agattgaccc tgcagtgact 1020 ccgcatggac ccctatacac tcacttcgga gagggccatc tctggcggag gctgaactct 1080 tggcacttcc gcccctccct gctgagccag agaagccctg gccattgtcc gtcactccga 1140 tagcctcacg gccaccctgt gcgtcccgcc ggtcgcccct tacccctggc tcgccccttc 1200 gcccttagat ggccagttct accaaagatg ggttccgctg catgatcccc gagacacccg 1260 cgccgggacc aagggtttca ttaaggtcac cttgtccgtg agggcgcgcg gggacctgcc 1320 ccctccaatg ctacccccgg ccccagggca ctgttcggac atcgagaagt gagccggggt 1380 gaggtgggga ggaggacatg gatccggggg tggccgtggg gcgcggataa ggggaggggc 1440 cgagatccca gtttctcccc ccccgctcgg tgccccctcc cctaggaacc tgctcctgcc 1500 gcgcggggtg cccgccgaga ggccatgggc gcggctccgc gtgcgcctgt accgcgccga 1560 ggggcttccc gcgctgcgcc tggggctgct gggcagcctg gtccgcgccc tgcacgacca 1620 gcgcgtcctg gtggagccct atgtgcgggt gtctttcctg gggcaggagg gcgagacgtc 1680 ggtgagcgcc gaggcggcgg cgcccgaatg gaacgagcag ctgagcttcg tggagctctt 1740 cccgccgctg acgcgcagcc tccgcctgca gctgcgggac gacgcgcccc tggtcgacgc 1800 ggcactcgct acgcacgtgc cggacctgag gcggatctcc catccgggcc gcgcggcggg 1860 gtttaaccct accttcggcc cggcctgggt gcccctctat ggctcgcccc ccggcgcggg 1920 gctccgggat agtcttcaag gtctcaacga aggcgttggc caaggcattt ggttccgcgg 1980 ccgccttctg ctggctgtgt ccatgcaggt gttggaaggg agagctgaac ctgagcctcc 2040 ccaggcccag caggggtcca cgttgtcccg gctcacccga aagaagaaaa agaaagccag 2100 aagggatcag accccaaagg cggttccgca gcacttggac gccagccccg gtgccgaggg 2160 gcctgagatc ccccgtgcca tggaggtgga ggtggaggag ctgctgccgc tgccagagaa 2220 tgtcctggcg ccctgtgaag atttcctgct tttcggtgtg ctcttcgagg ccaccatgat 2280 cgaccccacc gtggcctccc agcccatcag cttcgagatc tccattggtg tgtggcctag 2340 ccgaacccct gagtgccatt tcagacctta gaaccctgga aggggtgttg actttcagtc 2400 gcgcaggccg tctggaggag caattgggcc gagggtccag ggctggggag ggaactgagg 2460 gtgcagccgt ggaggctcag cctctgctgg gagccaggcc agaggaggag aaagaggagg 2520 aagaactggg gacccatgct cagcggcctg agcccatgga cggcagtggg ccatacttct 2580 gcttgcccct ctgtcactgc aagccatgca tgcatgtgtg gagttgctgg gaggaccaca 2640 cctggcgcct gcagagcagc aactgcgtgc gcaaagtggc cgagaggctg gaccaggggc 2700 tgcaggaggt tgagagactg cagcgcaagc cggggcctgg cgcctgtgca cagctcaagc 2760 aggcactgga agtactggtg gctgggagca gacagttttg ccacggtgcc gagcgcagga 2820 cgatgacccg gcccaatgcc ctggatcgat gccgagggaa actcctggtg cacagcctga 2880 accttttggc taagcaagga ctgcgacttc tacgcggcct gagacggcgc aatgtgcaaa 2940 agaaggtggc actggccaag aagctcctgg caaaactgcg ctttctggct gaggaggcac 3000 ccggggcagc ccctggtgag gtctgtgcca agctggagct cttcctgcgg ctgggcctgg 3060 gcaagcaagc caaggcctgc acctctgagc tgcccccgga tttgctgccc gagccctcag 3120 ccgggctgcc ctccagccta caccgggacg gtcctggagc agacgctgag ccctctgtgg 3180 gatgaactcc tggtatttga gcagttgatc gtggatggga ggagggagca cctgcaggag 3240 gagcctccat tagtgatcat caatgtattt gaccacaata agtttggccc ccccgtgttc 3300 ctgggcaggg cactggccgc cccaagggta aagctgatgg aggacccata ccaacgccca 3360 gagttgcagt tcttccccct gaggaaggga ccctgggcag ccggagagct cattgccgcc 3420 tttcaactca ttgaactaga ctacagtggc cgacttgagc cctcagtgcc cagtgaggtg 3480 gagccccagg atctggcacc cctggttgag ccccactctg gacgcctgtc ccttccaccc 3540 aacgtgtgcc cagtgctcag ggagttccgt gttgaggtgc tgttctgggg tcttagggga 3600 cttggtcgtg tgcatctgct cgaggtggag cagccccagg ttgtactgga ggtggctggg 3660 caaggtgtgg agtctgaggt cctggccagc taccgtgaga gccccaattt cactgagctt 3720 gtcaggcatc tgacagtggt cttcaaagac acagctcctc tcttccaccc ccaggacttg 3780 ccggagcagc cttacttgca gcctccactc agcatcttgg tgattgagcg ccgggccttt 3840 ggccacacag tccttgtggg ttcccacatt gtcccccaca tgctgcgatt cacatttcgg 3900 ggtcatgagg atcctcctga ggaggaagga gagatggagg agacagggga tatgatgccc 3960 aagggacctc aaggacagaa gtccctggat cccttcttgg ctgaagcggg tatatccaga 4020 cagctcctga agcacaactt tgatgaagat gaaatggatg atcctggaga ttcagatggg 4080 gtcaacctca tttctatggt tggggagatc caagaccagg gtgaggctga agtcaaaggc 4140 actgtgtccc caaaaaaagc agttgccacc ctgaagatct acaacaggtc cctgaaggaa 4200 gaatttaacc actttgaaga ctggctgaat gtgtttcctc tgtaccgagg gcaagggggc 4260 caggatggag gtggagaaga ggaaggatct ggacaccttg tgggcaagtt caagggctcc 4320 ttcctcattt accctgaatc agaggcagtg ttgttctctg agccccagat ctcccggggg 4380 atcccacaga accggcccat caagctcctg gtcagagtgt atgttgtaaa ggctaccaac 4440 ctggctcctg cagaccccaa tggcaaagca gacccttacg tggtggtgag cgctggccgg 4500 gagcggcagg acaccaagga acgctacatc cccaagcagc tcaaccccat ctttggagag 4560 atcctggagc taagcatctc tctcccagct gagacggagc tgacggtcgc cgtatttgat 4620 catgacctcg tgggttctga cgacctcatc ggggagaccc acattgatct ggaaaaccga 4680 ttctatagcc accacagagc aaactgtggg ctggcctccc agtatgaagt gtgggtccag 4740 cagggcccac aggagccatt ctgagtttct ggccaaacac attcaagctc acattccctt 4800 ttgtgtctcc agatcctatg atttcatgga aggggaccct cccacccacc gccactgcca 4860 accaagacat agctcagtgg tcaagacttg ggcttgggag tcgggatcct gtaacgaatg 4920 tcacttgacc gctttctttt tttatgaaac agtctcgctc tgtctcccag gttggagtgc 4980 agtggcacga tctcggctga ctgcaacctc cacctcctgg gttcaagcga ttctcctgcc 5040 tcagcctccc cagtagctgg gattacaggc gtgggccccc atgtccagct aatttttata 5100 ttttcgctct gtctcccagg ttggagtgca gtggcacgat ctcggctgac tgcaacctcc 5160 acctcctggg ttcaagcgat tctcctgcct cagcctcccc agtagctggg attacaggcg 5220 tgggccccca tgtccagcta atttttatat ttttagtaga gacagggttt caccatgttg 5280 tccaggctgg tcttgaaccc ctgacctcaa gtgatccacc cacctctgcc tcccaaagtg 5340 ctgggattac aggtgtgagc caccatgcca ggccctctta acctcttcaa gtctgttttc 5400 tcatctgcaa aacagaggta ataagatcag tatcttctta atggaagcac ctggactaca 5460 tttttttcat tcattgttat cataaatgag gactaacctg tctcccgttg ggagttttga 5520 acctagacct catgtcttca tgacgtcatc actgccccag gcccagctgt gtccctacac 5580 cagccccagc tgacgcatct tctttttctg cctgtagaga tggttacaat gcctggcgtg 5640 atgcattctg gccttcgcag atcctggcgg ggctgtgcca acgctgtggc ctccctgccc 5700 ctgaataccg agccggtgct gtcaaggtgg gcagcaaagt cttcctgaca ccaccggaga 5760 ccctgccccc agggatctct tcacatgtgg attgacatct ttcctcaaga tgtgcctgct 5820 ccacccccag ttgacatcaa gcctcggcag ccaatcagct atgagctcag agttgtcatc 5880 tggaacacgg aggatgtggt tctggatgac gagaatccac tcaccggaga gatgtcgagt 5940 gacatctatg tgaagagctg ggtgaagggg ttggagcatg acaagcagga gacagacgtt 6000 cacttcaact ccctgactgg ggaggggaac ttcaattggc gctttgtgtt ccgctttgac 6060 tacctgccca cggagcggga ggtgagcgtc tggcgcaggt ctggaccctt tgccctggag 6120 gaggcggagt tccggcagcc tgcagtgctg gtcctgcagg atccctggag ttgcagctac 6180 cagacatggt gcgtggggcc cggggccccg agctctgctc tgtgcagctg gcccgcaatg 6240 gggccgggcc gaggtgcaat ctgtttcgct gccgccgcct gaggggctgg tggccggtag 6300 tgaagctgaa ggaggcagag gacgtggagc gggaggcgca ggaggctcag gctggcaaga 6360 agaagcgaaa gcagaggagg aggaagggcc ggccagaaga cctggagttc acagacatgg 6420 gtggcaatgt gtacatcctc acgggcaagg tggaggcaga gtttgagctg ctgactgtgg 6480 aggaggccga gaaacggcca gtggggaagg ggcggaagca gccagagcct ctggagaaac 6540 ccagccgccc caaaacttcc ttcaactggt ttgtgaaccc gctgaagacc tttgtcttct 6600 tcatctggcg ccggtactgg cgcaccctgg tgctgctgct actggtgctg ctcaccgtct 6660 tcctcctcct ggtcttctac accatccctg gccagatcag ccaggtcatc ttccgtcccc 6720 tccacaagtg actctcgctg accttggaca ctcacccagg gtgccaaccc ttcaatgcct 6780 gctcctggaa gtctttctta cccatgtgag ctaccccaga gtctagtgct tcctctgaat 6840 aaacctatca cagcc 6855

TABLE LIII(f) Nucleotide sequence alignment of 121P1F1 v.1 (SEQ ID NO: 363) and 158P3D2 v.18 (SEQ ID NO: 364)

TABLE LIV(f) Peptide sequences of protein coded by 158P3D2 v.18 (SEQ ID NO: 365) MCKRRWHWPR SSWQNCAFWL RRHPGQPLVR SVPSWSSSCG WAWASKPRPA PLSCPRICCP 60 SPQPGCPPAY TGTVLEQTLS PLWDELLVFE QLIVDGRREH LQEEPPLVII NVFDHNKFGP 120 PVFLGRALAA PRVKLMEDPY QRPELQFFPL RKGPWAAGEL IAAFQLIELD YSGRLEPSVP 180 SEVEPQDLAP LVEPHSGRLS LPPNVCPVLR EFRVEVLFWG LRGLGRVHLL EVEQPQVVLE 240 VAGQGVESEV LASYRESPNF TELVRHLTVV FKDTAPLFHP QDLPEQPYLQ PPLSILVIER 300 RAFGHTVLVG SHIVPHMLRF TFRGHEDPPE EEGEMEETGD MMPKGPQGQK SLDPFLAEAG 360 ISRQLLKHNF DEDEMDDPGD SDGVNLISMV GEIQDQGEAE VKGTVSPKKA VATLKIYNRS 420 LKEEFNHFED WLNVFPLYRG QGGQDGGGEE EGSGHLVGKF KGSFLIYPES EAVLFSEPQI 480 SRGIPQNRPI KLLVRVYVVK ATNLAPADPN GKADPYVVVS AGRERQDTKE RYIPKQLNPI 540 FGEILELSIS LPAETELTVA VFDHDLVGSD DLIGETHIDL ENRFYSHHRA NCGLASQYEV 600 WVQQGPQEPF 610

TABLE LV(f) Amino acid sequence alignment of 121P1F1 v.1 and 158P3D2 v.18 No significant similarity. 

1. An isolated polynucleotide that encodes a 158P3D2 protein, wherein the polynucleotide is selected from the group consisting of: (a) a polynucleotide comprising the sequence of SEQ ID NO:2, from nucleotide residue numbers 1 through 849, wherein the nucleotide residue at 150 is G; (b) a polynucleotide comprising the sequence of SEQ ID NO:2, from nucleotide residue numbers 849 through 1835, wherein the nucleotide residue at 1022 is A; (c) a polynucleotide comprising the sequence of SEQ ID NO:2, from nucleotide residue numbers 849 through 1835, wherein the nucleotide residue at 1148 is A; (d) a polynucleotide comprising the sequence of SEQ ID NO:2, from nucleotide residue numbers 849 through 1835, wherein the nucleotide residue at 1691 is T; and (e) a polynucleotide comprising the sequence of SEQ ID NO:2, from nucleotide residue numbers 849 through 1835, wherein the nucleotide residue at 1692 is G.
 2. An isolated polynucleotide that is the full complement to the polynucleotide of claim
 1. 3. The polynucleotide of claim 1 that encodes the polypeptide sequence shown in SEQ ID NO:3 wherein S58R, K281N, or T282A.
 4. A recombinant expression vector comprising the polynucleotide of claim
 1. 5. An isolated host cell that contains the expression vector of claim
 4. 6. A process for producing a 158P3D2 protein comprising culturing the host cell of claim 5 under conditions sufficient for the production of the 158P3D2 protein.
 7. The process of claim 6, further comprising recovering the 158P3D2 protein so produced.
 8. The process of claim 6, wherein the protein is recovered using chromatography. 